Ureaplasma vaccine and antibody for prevention and treatment of human, animal and cell culture infection

ABSTRACT

The present invention encompasses methods and compositions for  Ureaplasma  infection prevention and/or treatment. In specific cases, the invention concerns vaccines for  Ureaplasma , including DNA vaccines. In certain embodiments, the invention regards vaccines directed towards the multiple-banded antigen(s) of  Ureaplasma.

This application is a national phase application under 35 U.S.C. §371that claims priority to International Application No. PCT/US2012/035779filed Apr. 30, 2012 which claims priority to U.S. ProvisionalApplication Ser. No. 61/480,639, filed Apr. 29, 2011, both of which areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present invention generally concerns the fields of immunology, cellbiology, molecular biology, infectious disease, and medicine. Inspecific embodiments, the present invention concerns immunologicalcompositions and related methods for Ureaplasma, including vaccines.

BACKGROUND OF THE INVENTION

There are up to 7 species of Ureaplasma, The two species associated withhuman infection are Ureaplasma parvum and Ureaplasma urealyticum. Allspecies within the genus Ureaplasma, family Mycoplasmataceae. They areprokaryotes devoid of a cell wall and hence insensitive to penicillinand gram stain. They are small (0.1-0.85 um) and best visualized inbroth culture by dark-field or phase-contrast microscopy, but itspleomorphic nature makes it difficult to identify in medium. Thus,organisms typical colonies are recognized on solid medium (7-30 um) andis the sine qua non for identification. (Taylor-Robinson and Gourley,1984)

Ureaplasma need urea for growth even in highly complex media and producethe enzyme urease which allows the organism to metabolize urea.(Pollack, 1986) They do not synthesize folic acid and as such are notsusceptible to sulfonamides or trimethoprim. Ureaplasma producehemolysin. (Furness, 1973; Shepar and Masover, 1979) Ureaplasma appearto attach to a variety of host cells via unique mechanisms and theninvade the host cell. (Busolo et al., 1984; masover et al., 1977;Robertson et al., 1991; Saada et al., 1991; Shepard and Masover, 1979;Torres-Morquecho et al., 2010) This has been associated with cellapoptosis (Li et al., 2002) and increased inflammatory cytokines.Several have reported that Hela (McGarrity and Kotani, 1986; Smith etal., 1994) or A549 (Torres-Morquecho et al., 2010) cells can be used tostudy this attachment.

Serologic and genomic relationships among the established andunspeciated Ureaplasma species and serovars isolated from various hostscan be summarized as follows. (human) is separated into two genomicclusters (parvum and urealyticum). Ureaplasma diversum (bovine) hasthree serologic clusters that identify all U. diversum strains. Thenonhuman primate strains form four serologic groups, and each serogroupis composed of strains isolated from primates belonging to one of fourdistinct zoologic primate families. The ovine-caprine strains have twoserologic clusters. Canine strains form four serologic clusters butserovars 1 and 2 are closely related by DNA homology. Avian strainsbelong to one serogroup with two genomic clusters. (Barile M F, PediatrInfect Dis 1986 5 (6 Suppl):S296-9).

U. urealyticum and U. parvum have at least 14 serotypes defined byserologic and biologic characteristics among its numerous strains. Theseserotypes have recently been subdivided into two biovar: U. urealyticumor group 2 (serotypes 2,4,5,7,8,9,10,11,12,13); U. parvum or group 1(serotypes 1,3,6,14). (Robertson et al., 2001) The genome size of thevarious strains appears to vary widely and corresponds to the twoserovar clusters. The genome size of cluster group 1 is about 760 kb,while group 2 ranges from 880 to 1,140 kb. (Robertson et al., 1990)Serovar identification can be accomplished by serology (Roberson andStemke, 1982), immunofluorescence (Roberson and Stemke, 1982), and ELISA(Brown et al., 1981; Horotzitz et al., 1995). The latter is least laborintensive and has been reproduced (Turunen et al., 1982; Wiley andQuinn, 1984). It may be difficult to detect all serovar because ofvariable growth rates (Stemke and Robertson, 1985), and multiple serovarper specimen (Quinn, 1986).

The most sensitive method of isolating Ureaplasma consists of specimeninoculation into liquid medium and subculture to agar. (Robertson, 1978;Taylor-Robinson et al., 1967; Taylor-Robinson and Gourley, 1984;Taylor-Robinson, 1989) Colonies sometimes fail to develop when aspecimen is plated directly on agar. In liquid medium, organisms aredetected by their urease activity. Small colonies occur on agargenerally due to lack of the classical fried-egg appearance, butimproved medium has increased colony size, and manganous sulfate orcalcium chloride, both sensitive indicators of ammonia, result in darkbrown Ureaplasma colonies. (Shepard and Masover, 1979; Taylor-Robinsonand Gourlay, 1984; Taylor-Robinson, 1989)

The multiple banded antigen (MBA) gene is present in all serovar ofUreaplasma (Teng et al., 1994). This gene appears to play a significantrole in the organism's virulence (Kong et al., 1999), and the gene's 5′regions are markers of biovar specificity and diversity (Teng et al.,1994). This region can not only be used to differentiate U. parvum fromU. urealyticum, it indicates that there may be 5 MBA genotypes of the U.urealyticum species: A (serovars 2,5,8), B (serovar 10), C (serovars4,12,13), D (serovar 9), E (serovars 7,11). The MBA gene has been clonedand sequenced. (Zheng et al., 1994) The MBA gene consists of a conservedsection encoding both a signal peptide and a membrane anchor, and avariable section encoding a number of uniform repeating units.(Zimmerman et al., 2011) Thus, selection of that portion of the MBA genethat codes for a constant region is an excellent target for vaccine orantibody development, in specific embodiments of the invention. The MBAgene for serotype 6 was selected for initial development of the vaccineof the invention, because it is a frequently isolated clinical serotype.(Vancutsem et al., 2008) The MBA appears significant in attachment ofthe organism. (Monecke et al., 2003; Torres-Morquecho et al., 2010) MBAalso appears to activate NF-kappaB through TLR1, TLR2 and TLR6 andinduce tumour necrosis factor-alpha (TNFalpha). (Shimizu et al., 2008)The number of MBA variants in vivo is inversely related to thedevelopment of clinical inflammation. (Knox et al., 2010)

Simple and rapid methods of Ureaplasma identification have beendeveloped, but now only confirm culture. A solid phase enzymeimmunoassay is not reliable. (Taylor-Ronbinson, 1989) A whole chromosomeDNA probe was insensitive (especially <10³ ccu/ml) and was positive forculture-negative specimens. (Roberts et al., 1987) A PCR for Ureaplasmaappears a very good indicator of infection. (Blanchard and Gautier,1990; Willoughby et al., 1990) and clinical evaluations have confirmedthis (Abele-Horn et al., 1996; Blanchard et al., 1993; Cunliffe et al.,1996), but commercial kits are not yet readily available.

Clinical Significance of Organism: Ureaplasma is a sexually transmittedinfection associated with a broad range of clinical diseases in men andwomen including non-gonococcal urethritis, urinary stone formation,suppurative arthritis, and infertility. In men, it causes non-gonococcalurethritis and prostatitis. In women it causes pelvic inflammatorydisease, recurrent abortion, chorioamnionitis, stillbirths, prematurebirth, low birth weight, and postpartum endometritis. In newborn babiesit is associated with several diseases including pneumonia, sepsis,meningitis, osteomyelitis, death, intraventricular hemorrhage,periventricular leukomalacia, necrotizing enterocolitis (Pediatr. Res.2011 May; 69 (5 Pt 1):442-7, and chronic lung disease. (O'Leary, 1990;Pinna et al., 2006; Waites et al., 2005) However, there is variableoccurrence of these diseases in patients colonized with this organism.(Krause and Taylor-Robinson, 1992) The variable development of diseasein colonized patients indicates a virulence factor among pathogenicstrains, or antibody, variability or both.

Data on the genital tract colonization of non-pregnant women arelimited, but appear high in sex workers (44%), STD clinic clients (40%)(Kong et al., 1999), family planning clinic (43%) (Domingues et al.,2002), symptomatic (48%) and asymptomatic (22%) STD clinic patients(Gupta et al., 2008), In addition Ureaplasma has been isolated from thesemen of 12% (9% urealyticum, 3% parvum) of all men with infertilitycompared to 3% (2% parvum, 1% urealyticum) of those who are fertile(Zeighami et al., 2009).

Colonization of the lower genital tract with Ureaplasma in pregnantwomen is very common varying from 44 to 88%. (Carey et al., 1991;Cassell et al., 1993; Eschenbach, 1993; Kundsin et al., 1996; Luton etal., 1994) Colonization of the lower genital tract with serotype 3 or 6Ureaplasma is associated with an MBA antibody response to the variableregion of these Ureaplasma serotypes in 51% of women while 15% of womenwho were not-colonized with these organisms demonstrated the sameantibody. (Vancutsem et al., 2008)

Colonization of the upper genital tract or amniotic fluid withUreaplasma in pregnant women appears to be strongly associated withadverse pregnancy outcomes including spontaneous miscarriage, pre-termlabor, pre-labor rupture of membranes, and post-partum endometritis andmay occur without microscopic or clinical signs of inflammation.(Andrews et al., 1995; Cassell et al., 1983; Font et al., 1995; Gray etal., 1992; Hazan et al., 1995; Horowitz et al., 1995; Kundsin et al.,1996)

The inventors recently completed a prospective case-control study todetermine if Ureaplasma colonization or infection of the placenta isassociated with an increase in adverse pregnancy outcome, in particularpremature birth. (Okunola et al., 2006; Okunola et al., 2007) Twohundred fifty-two women who gave birth at three Baylor affiliatedhospitals (St Luke's Episcopal Hospital, Methodist Hospital, and BenTaub General Hospital) during an 18 month period participated. Thesewomen were composed of 3 groups: 58 gave birth to premature infantsbetween 20 and 30 wks gestation; 27 developed perinatal complications(prolonged rupture of membranes >18 hours, premature rupture ofmembranes, maternal fever >100.4° F., or clinical chorioamnionitis orendometritis) and gave birth to term infants; 167 had no perinatalcomplications and gave birth to term infants. Over 40% of those womenwho gave birth to premature infants (p<0.0001) or who had perinatalcomplications with a term birth (p<0.004), had placental colonization orinfection with Ureaplasma, compared to term births without perinatalcomplication who had a <15% Ureaplasma placental colonization orinfection. No maternal demographic, medical, surgical, or pregnancyfactors appear to predict Ureaplasma infection or colonization of theplacenta. Of the 58 preterm infants, (Molina et al., 2010) 23 placentaswere culture positive for Ureaplasma (40%). Infants whose placenta werepositive were not different then those who were negative, in eithergestation (26±2.4 vs 26±2.1 wks), birth weight (884±278 vs 890±401),male sex (44% vs 54%), race (38% vs 31%), and prenatal factors. 70% ofthe Ureaplasma were biovar 1, and of those all were either serotypes 3,6, or 14. Of infants who survived to 36 wks corrected gestational age(CGA), BPD developed in 69% with Ureaplasma in their placenta comparedto 37% of those with a negative culture (p=0.062). Of all infants, deathor BPD resulted by 36 wks CGA in 78% with Ureaplasma in their placentacompared to 51% of those with a negative culture (p=0.054). Antenatalexposure of the fetus to Ureaplasma may increase the risk of BPD ordeath. Strategies to prevent Ureaplasma placenta colonization maydecrease premature birth and its complications.

To determine those women at risk for placenta colonization, theinventors recently completed a prospective study (Weisman et al., 2009)of 290 women evaluating Ureaplasma vaginal colonization, and thefollowing was observed: 44% of women at 16 wks gestation had vaginalUreaplasma colonization; colonization did not change significantlythroughout gestation; 32% of all colonized women developed placentalUreaplasma infection (12% of all); all women with placental Ureaplasmainfection had vaginal colonization at 16 wks gestation. In pretermbirths: 67% had vaginal colonization; this did not change throughoutgestation; 62% of colonized women developed placental Ureaplasmainfection (42% of all). Vaginal colonization at 16 wks gestation is anearly marker for those at risk of poor pregnancy outcome and potentialtarget intervention, in certain cases of the invention. Although otherconditions (e.g. other infections, anatomic abnormalities, endocrinedisorders, maternal medical conditions, etc.) may contribute to poorpregnancy outcome, Ureaplasma colonization of the placenta appears asignificant association. If those at risk for poor outcome can beidentified early, intervention strategies including antibiotics or morelikely vaccines could provide protection from Ureaplasma and adversepregnancy outcomes.

It has been proposed that Ureaplasma should be eradicated from theurogenital tracts of women and their partners. (Kundsin et al., 1996)Ureaplasma is not susceptible in vitro to penicillins, sulfonamides,trimethoprim, aminoglycosides, and clindamycin, but are generally (about90%) susceptible in-vitro to tetracyclines, and variably to macrolides(e.g. erythromycin). (Cassell et al., 1993) The inventors have confirmedin recent studies the variable susceptibility of Ureaplasma toerythromycin in vitro. In view of the high colonization rate and sexualtransmission rates of Ureaplasma, it is unlikely that such strategieswill be effective in its eradication. In addition, this organism hasbeen observed to persist in the genital tract despite antibiotictreatment. In couples attending an infertility clinic this organismpersisted in the genital tract despite antibiotic treatment. (Hipp etal., 1983) Routine use of intraoperative prophylactic-antimicrobialtherapy at Cesarean delivery did not effect Ureaplasma colonization ofthe chorioamnion at delivery. (Andrews et al., 1995) Macrolides(Eschenbach et al., 1991; Mazor et al., 1993; Romero et al., 1993) havenot been reliable in eradicating genital tract Ureaplasma or adverseperinatal outcomes in two randomized controlled trials. Although newerantibiotics such as glycylcyclines (Kenny and Cartwright, 1994) andquinolones (Kenny and Cartwright, 1996) may prove more effective, theirsafety and efficacy during pregnancy are unproven.

It has been suggested, but not demonstrated, that lack of specificantibody may be critical for preventing Ureaplasma infection, becausespecific protein antibody may inhibit growth in vitro. (Cassell et al.,1993) Hypogammaglobulinemic patients have an increased susceptibility toUreaplasma. (Taylor-Robinson et al., 1986) Serological studies ofhypogammaglobulinemic patients (Volger et al., 1985), pre-term infants(Quinn et al., 1983), and women with recurrent spontaneous abortions(Quinn et al., 1983) support this concept. Increased susceptibility ofinfants of <30 wks gestational age to Ureaplasma induced respiratorydisease may be related to their hypogammaglobulinemia (Ballow et al.,1986) or to their lack of specific antibody (Cassell et al., 1988;Cassell et al., 1988).

It has been suggested, but not demonstrated, that monoclonal antibodiesto specific protein antigens of Ureaplasma can inhibit growth of theseorganisms in vitro and indicates that specific antibody may be importantfor host defense. (Watson et al., 1990) There is a long-felt need in theart to provide useful methods and reagents for Ureaplasma vaccines andmethods and compositions to prevent or treat Ureaplasma infection.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to immunological methods andcompositions for Ureaplasma, including vaccines and antibodies forprevention and/or treatment of mammalian infection, including, forexample, a DNA vaccine for its related antibodies. In particularembodiments, the compositions are useful to prevent infection and alsoto reduce the deleterious effects of infection once the individual isinfected. In specific embodiments, the composition may be employed for afemale or a male or both. The compositions may be utilized in adults,adolescents, children, or infants. In specific cases, the composition isdelivered to an individual prior to the onset of becoming sexuallyactive, including becoming sexually active for the first time. The typeof sexual activity may be of any kind. An adolescent may be vaccinatedat or about the time of onset of puberty. In certain cases, a female isvaccinated prior to pregnancy, while in other cases a female isvaccinated during pregnancy. In some embodiments of the invention, anindividual susceptible to or having immune deficiency syndromes that areeither congenital (e.g. agammaglubulinemia) or acquired (e.g. patientswith cancer receiving or not receiving therapy) are administered methodsand compositions of the invention. In specific embodiments, anindividual in early childhood is treated with at least some aspects ofthe invention, including antibody or vaccine compositions. In specificcases, an individual after the diagnosis of cancer or the diagnosis ofimmune deficiency is provided methods and/or compositions of theinvention. In certain cases, a male or a female is vaccinated whensexually active or prior to being sexually active.

An individual that is administered compositions and methods of theinvention may be susceptible to having Ureaplasma infection, may besuspected of having Ureaplasma infection, or may be known to haveUreaplasma infection or high risk for infection. In at least certaininstances that the individual is known to have Ureaplasma infection, theindividual may also be administered another therapy for Ureaplasma,including certain antibiotics, for example.

Ureaplasma infection during pregnancy and delivery has been suggested tocause abnormal brain development in the baby in a few clinical studiesand one animal study. The role of Ureaplasma infection during pregnancyon brain development abnormalities is encompassed in the invention. Itappears to not only affect the long term behavior of premature babies,but could have a role in other brain conditions associated withinflammation including brain injury due to lack of oxygen, bloodinfection, brain infection, and severe jaundice in the newborn, andseizures, cerebral palsy, autism, and attention deficit hyperactivity inpediatrics. The present disclosure encompasses a mouse model andaddresses the impact of Ureaplasma infection during pregnancy on braininflammation and behavior of the baby and also encompasses methods andcompositions for preventing brain development abnormalities in a fetusor infant.

Ureaplasma infection's role in brain development abnormalities is animportant area of investigation, because it appears to not only affectthe development outcome of preterm infants, but in some cases of theinvention it appears to have a role in other brain conditions associatedwith inflammation, including hypoxic ischemic perinatal brain injury,sepsis, meningitis, and hyperbilirubinemia in the neonate, and seizures,cerebral palsy, autism spectrum disorders, and attention deficithyperactivity disorders in pediatrics. The present invention includesthe impact of perinatal Ureaplasma infection and inflammation on braindevelopment.

The present invention includes a murine model of antenatal Ureaplasmachorioamnionitis, in certain cases. In some embodiments, it includesdetermination of the effect of Ureaplasma chorioamnionitis on braindevelopment in the suckling mouse, including behavior and memory, brainpathology and structure, and molecular signals. In certain embodiments,the present invention includes determination of antenatal maternaladministration of an Ureaplasma recombinant DNA (rDNA) vaccine, proteinvaccine, or monoclonal antibody that affects Ureaplasma related changesin brain development.

In some embodiments of the invention, there is an immunologicalcomposition (such as an antibody) that immunologically reacts with amultiple-banded antigen of Ureaplasma, said composition comprised in apharmacologically acceptable excipient. In specific embodiments, thecomposition is further defined as a vaccine, including a DNA, protein,or antigen vaccine. In specific embodiments, the vaccine comprises oneor more DNA polynucleotides, protein, or antigen. In certain cases, thevaccine comprises monoclonal or polyclonal antibodies.

Certain embodiments include diagnosis of Ureaplasma infection, forexample by PCR. Specific embodiments utilize antibodies of the inventionfor Ureaplasma detection, such as from an individual or from a culture.

In particular embodiments, the antibodies of the invention are employedfor cell culture and media contamination applications. Exemplary cellculture lines and media are well known in the art (Hassan M, et al. JBasic Microbiol. 2010; Harasawa R, et al. Res Microbiol. 1993.; Kong F,et al. Appl Environ Microbiol. 2001.; Wang H, et al. Appl EnvironMicrobiol. 2004.; Sung H, et al. J. Microbiol. 2006. Johansson K E, etal. Molecular and Cellular Probes. 1990.; Teyssou R, et al. Molecularand Cellular Probes. 1993). In at least some specific aspects, theantibody directly kills the organism in media without complement orneutrophils or macrophages.

In some embodiments of the invention, there is a DNA vaccine comprisinga polynucleotide encoding part or all of a Ureaplasma antigen. Inspecific embodiments, the antigen is urease, UU376 gene product,virulence gene product, or urea transporter, or wherein thepolynucleotide comprises MBA N-terminal paralogs, 16S rRNA, the areaupstream of the Urease A gene, the Urease A-Urease B spacer, the UreaseB-Urease C spacer, or the 16S-23S rRNA intergenic spacer region. Incertain aspects, the vaccine is further defined as a DNA vaccinecomprising a polynucleotide encoding at least one multiple-bandedUreaplasma antigen.

In nucleic acid vaccine embodiments, the polynucleotide may be furtherdefined as follows: a) comprises a strong viral promoter; b) comprisesMason-Pfizer monkey virus (MPV)-CTE with or without rev; c) comprisesIntron A or an intron from SV40 or Raucous sarcoma; d) strongpolyadenylation/transcriptional termination signal; e) expresses themultiple binding proteins from more than one species, biovar, serotypeor strain of Ureaplasma; f) comprises codons for pathogenic mRNA; g)comprises an immune enhancer (such as from human granulocyte-macrophagecolony-stimulating factor); h) comprises a N-terminal ubiquitin signal;i) comprises strings of minigenes (or MHC class I epitopes from)different pathogens or oligonucleotides (for example, wherein thestrings of MHC class I epitopes from different pathogens oroligonucleotides comprise a CpG motif); j) comprises a TH epitope; or k)a combination thereof.

In certain aspects of the vaccine embodiments, the vaccine may befurther defined as comprising two Ureaplasma antigens. In specificcases, a DNA vaccine is further defined as comprising ANNATPG in frontof the start codon.

In particular embodiments, there is a vaccine that immunologicallyreacts with a multiple-banded antigen of Ureaplasma, said vaccinecomprised in a pharmacologically acceptable excipient. In a specificembodiment, the vaccine comprises a peptide or polypeptide of themultiple-banded antigen. In specific aspects, the vaccine comprises anantibody that immunologically reacts with the multiple-banded antigen.

In one embodiment of the invention, there is a kit comprising a vaccineof the invention housed in a suitable container.

In one embodiment of the invention, there is a method of preventingUreaplasma infection in an individual or reducing symptoms of Ureaplasmainfection in an individual, comprising the step of delivering atherapeutically effective amount of an antibody or vaccine of theinvention to the individual. In specific embodiments, the individual isa human, cow, female, male, etc. In some cases, the individual is afemale or male prior to a first sexual activity or the individual is afemale prior to pregnancy. The vaccine may be delivered to a pregnantfemale. The individual may be an infant, child, or adolescent. In someembodiments, there is a method of preventing Ureaplasma infection in acell media, comprising the step of delivering to the media an effectiveamount of antibodies that recognize the conserved region of Ureaplasmamultiple-banded antigen or the 5′ end of the multiple-banded antigen.

In certain embodiments, the antibodies or vaccine are delivered byinjection, such as intramuscular, intravenous, subcutaneous,intraperitoneal, by Gene Gun, by pneumatic injection, or it comprisesliposomes. In specific cases, when the vaccine comprises DNA the GeneGun comprises delivery of DNA coated gold or tungsten beads viaepidermal delivery. In certain cases, when the vaccine comprises DNA thepneumatic injection is via epidermal delivery. Particular aspects of theinvention further comprising multiple deliveries to the individual, suchas deliveries being separated by years, months, weeks, or days, forexample. In specific cases, the multiple deliveries are separated by onemonth or more. In specific cases, the multiple deliveries are separatedby between two and ten years. In some embodiments, the vaccine orantibody is delivered in the amniotic cavity or vaginally, for example.

In some embodiments, there are antibodies that immunologically reactwith a conserved region of Ureaplasma multiple-banded antigen or the 5′end of the multiple-banded antigen. In certain embodiments, there is amethod of preventing Ureaplasma infection in an individual or reducingsymptoms of Ureaplasma infection in an individual, comprising the stepof delivering to the individual a therapeutically effective amount ofantibodies that recognize the conserved region of Ureaplasmamultiple-banded antigen or the 5′ end of the multiple-banded antigen.

Thus, in embodiments of the invention there is cloning and expression ofa conserved section of Ureaplasma multiple banded antigen gene, such asin the exemplary pVAX1 vector. The data provided herein includesefficacy demonstrated in-vitro (IgG bacterial binding, IgA bacterialbinding, bacterial killing) and in-vivo (animal protection). Thisexemplary work demonstrated that this vaccine through its antibodiesand, optionally, other factors was effective in binding Ureaplasmain-vitro, neutralizing (killing) Ureaplasma in-vitro independent ofother immune factors (complement and neutrophils), and providingprotection (decreased mortality and bacteremia) to animals infected withUreaplasma. In embodiments of the invention, vaccine-related antibodieshave application in the prevention and treatment of human infection,prevention and treatment in animal infection, and prevention andtreatment of media that has been contaminated with Ureaplasma.

Also encompassed in the invention are optimized vaccine delivery, dose,and schedule methods, and the immunologic response to the vaccine andrelated antibodies is evaluated herein.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Serum IgA level in vaccinated mice.

FIG. 2. Serum level of IgM in vaccinated mice

FIG. 3. Serum level of IgG subclasses in vaccinated mice

FIG. 4. Serum level of pathogen specific IgG in vaccinated mice. Eachdata point contains serum from 3 animals and repeated 3 times.

FIG. 5. Serum level of pathogen specific IgA in vaccinated mice. Eachdata point contains serum from 3 animals and repeated 3 times.

FIG. 6. In vitro bacterial killing assay of serotype A (U. diversum), 1,(U. parvum) and 8 (U. urealyticum) with serum from mice immunized (IMS)with Ureaplasma DNA vaccine serotype 6 (U. parvum) and serum from normalmice (NMS). Column 1, 5 & 6. Ureaplasma (Column 1 is Serotype A; Column5 is Serotype 1; Column 6 is Serotype 8)+IMS. Column 3, 7 and 8 are 10Bonly. Column 2 and 4 are Serotype A+NMS. Column 9 is Serotype 1+NMS.Column 10 is Serotype 8+NMS.

FIG. 7. In vitro bacterial killing assay of Ureaplasma diversum serotypeA with serum from mouse immunized (IMS) with Ureaplasma DNA vaccineserotype 1 and 6 or normal mouse serum (NMS). All wells contain 10Bbroth+Ureaplasma except Column 3. Column 1: IMS. Column 2: NMS. Column 310B only. Column 4 and 5: 10 B broth.

FIG. 8. Animal survival rate of the vaccine and nonvaccine groupsagainst Ureaplasma infection with either serotypes 6 (U. parvum), 8 (U.urealyticum), or 14 (U. parvum).

FIG. 9. Animal survival rate of the vaccine and nonvaccine groupsagainst Ureaplasma serotype 14 infection.

FIG. 10. Animal survival rate of the vaccine and nonvaccine groupsagainst Ureaplasma serotype 6 infection.

FIG. 11. Animal survival rate of the vaccine and nonvaccine groupsagainst Ureaplasma serotype 8 infection.

FIG. 12. Animal survival rate of the vaccine (serotype 1&6) andnonvaccine groups against Ureaplasma serotype 14 infection.

DETAILED DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention disclosed inthis Application without departing from the scope and spirit of theinvention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternative are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.”

As used herein, the terms “effective amount” or “therapeuticallyeffective amount” are interchangeable and refer to an amount thatresults in an improvement or remediation of at least one symptom of thedisease or condition. Those of skill in the art understand that theeffective amount may improve the patient's or subject's condition, butmay not be a complete cure of the disease and/or condition.

The term “preventing” as used herein refers to minimizing, reducing orsuppressing the risk of developing a disease state or parametersrelating to the disease state or progression or other abnormal ordeleterious conditions.

The terms “treating” and “treatment” as used herein refer toadministering to a subject a therapeutically effective amount of acomposition so that the subject has an improvement in the disease orcondition. The improvement is any observable or measurable improvement.Thus, one of skill in the art realizes that a treatment may improve thepatient's condition, but may not be a complete cure of the disease.Treating may also comprise treating subjects at risk of developing adisease and/or condition.

I. General Embodiments of the Invention

Certain embodiments of the invention include compositions useful forpreventing and/or treating Ureaplasma in a mammal, including a human,cow, dog, cat, horse, pig, goat, or sheep, or a bird, for example.Certain aspects include compositions useful for cell cultures of humanor animal tissues or culture media for medical, microbiological,pharmaceutical, etc. use for treating Ureaplasma contamination or forprevention of such contamination. The compositions may includeantibodies, proteins, peptides, nucleic acid expression vectors, and soforth. In some cases, the compositions may be considered vaccines. In atleast some embodiments of the invention, an individual is provided themethods and/or compositions in order to prevent infection by Ureaplasma,and this individual may or may not be pregnant. In the event that afemale or male individual is inoculated with a composition of theinvention, the female or male may then be prevented from havingdeleterious effects upon challenge with Ureaplasma. In at least certaincases, a fetus in a current or later pregnancy of a female is protectedfrom the deleterious effects of Ureaplasma. There may be more than onefetuses simultaneously protected or successively protected.

Furthermore, Ureaplasma infection during pregnancy and delivery has beensuggested to cause abnormal brain development in the baby in a fewclinical studies and one animal study. The present invention includesembodiments wherein the role of Ureaplasma infection during pregnancy onbrain development abnormalities is encompassed. In specific embodiments,it affects not only the long term behavior of premature babies but in atleast some cases has a role in other brain conditions associated withinflammation including brain injury due to lack of oxygen, bloodinfection, brain infection, and severe jaundice in the newborn,seizures, cerebral palsy, autism, and attention deficit hyperactivity inpediatrics. In embodiments of the invention, a mouse model is utilizedto demonstrate the impact of Ureaplasma infection during pregnancy onbrain inflammation and behavior of the baby.

The compositions and methods of the invention are useful fornon-gonococcal urethritis, urinary stone formation, suppurativearthritis, infertility, prostatitis, pelvic inflammatory disease,recurrent abortion, chorioamnionitis, stillbirths, premature birth, lowbirth weight, postpartum endometritis, pneumonia, sepsis, meningitis,osteomyelitis, death, intraventricular hemorrhage, periventricularleukomalacia, necrotizing enterocolitis, and chronic lung disease, inparticular aspects.

II. Multiple-Banded Antigens of Ureaplasma

In some embodiments of the invention, the immunological composition,including a vaccine, utilizes the multiple-banded antigen as itsantigen.

There are fourteen acknowledged serovars of Ureaplasma that can bedivided into two clusters of biovars: Biovar 1 (or parvum biovar) havingserovars 1, 3, 6, and 14; and Biovar 2 (or urealyticum or T960 biovar)having 10 serovars of 2, 4, 5, 7, 8, 9, 10, 11, 2, and 13. Members ofBiovar 1 and Biovar 2 can be distinguished at least by DNA-DNAhybridization, restriction fragment length polymorphism, 1D and 2D gelelectrophoresis, genomic sizes, and PCR amplification of certain genes.The different serovars each have a distinct antigen that, in some casesof the invention, are utilized as a target for the immunologicalcomposition. There are predominant antigens recognizable in patientsinfected with Ureaplasma (Watson et al., 1990; see also Teng et al.,1994; Zheng et al., 1995; Kong et al., 1999a; 1999b; nd Kong et al.,2000), and these are referred to as multiple-banded antigens.

Exemplary multiple-banded antigens of Ureaplasma include at least thefollowing from different serovars, denoted by their GenBank® sequences,all of which are incorporated by reference herein: AAD09745.2;AAD09744.2; AAD09743.2; AAD02701.2; AAD02700.2; AAD02699.2; AAD02698.2;AAD02697.2; AAD02696.2; AAD02695.2; AAD02694.2; AAD02693.2; AAD02692.2;AAD00075.1; AAC41437.1; AAD00077.1; AAD00076.1; AAB38978.1; AAD19956.1;AAD19955.1; AAD19954.1; AAD19953.1; AAD19952.1; AAD19951.1; AAD19950.1;AAD19949.1; AAD19948.1; AAD19947.1; AAD19946.1; NP_(—)078209.1;YP_(—)002284809.1; YP_(—)002284808.1; YP_(—)002284599.1;YP_(—)002284567.1; YP_(—)002284811.1; YP_(—)002284585.1;YP_(—)001752457.1; ACI60346.1; ACI60338.1; ACI60127.1; ACI60016.1;ACI59928.1; ACI59882.1; ACA32903.1; AAF61146.1; AAF61145.1; AAF30784.1;ABU75287.1; AAT79416.1; AAT79415.1; AAT79414.1; AAT79413.1; AAT79412.1;AAT79411.1; ZP_(—)03772473.1; ZP_(—)03772432.1; ZP_(—)03772428.1;ZP_(—)03772407.1; ZP_(—)03772313.1; ZP_(—)03772151.1; ZP_(—)03003758.1;ZP_(—)02997126.1; ZP_(—)02570847.2; ZP_(—)02553955.2; ZP_(—)02555017.2;ZP_(—)02691486.2; ZP_(—)02691471.2; ZP_(—)02690312.2; ZP_(—)02691487.1;ZP_(—)02691469.1; ZP_(—)02690307.1; ZP_(—)02690299.1; ZP_(—)02570851.1;ZP_(—)02570848.1; ZP_(—)02555874.1; ZP_(—)02555020.1; ZP_(—)02555016.1;ZP_(—)02555015.1; ZP_(—)02555013.1; ZP_(—)02553953.1; ZP_(—)03771933.1;ZP_(—)03771930.1; ZP_(—)03771929.1; ZP_(—)03771924.1; ZP_(—)03771712.1;ZP_(—)03771427.1; ZP_(—)03771410.1; ZP_(—)03771378.1; ZP_(—)03771338.1;ZP_(—)03771299.1; ZP_(—)03771272.1; ZP_(—)03771271.1; EEH02496.1;EEH02495.1; EEH02491.1; EEH02279.1; EEH01994.1; EEH01977.1; EEH01945.1;EEH01905.1; EEH01866.1; EEH01840.1; EEH01837.1; EEH01836.1; EEH01707.1;EEH01666.1; EEH01662.1; EEH01641.1; EEH01547.1; EEH01385.1;ZP_(—)03206353.1; EDY74356.1; ZP_(—)03079858.1; ZP_(—)03079727.1;EDX53837.1; EDX53694.1; EDX53543.1; EDX53525.1; EDX53195.1;ZP_(—)02558219.2; ZP_(—)02558216.1; ZP_(—)02558215.1; EDU67250.1;EDU67198.1; EDU56636.1; EDU56624.1; EDU56617.1; EDU56613.1; EDU19480.1;EDU19358.1; EDU06306.1; EDU06277.1; EDU06259.1; EDU06258.1; EDU06213.1;EDT87551.1; EDT87494.1; EDT48735.1; EDT48714.1; EDT48712.1; EDT48706.1;and/or EDT48704.1.

In certain embodiments of the invention, an immunological composition,such as a vaccine, is effective by being able to immunologically reactwith a variety of multiple-banded antigens, and in some embodiments theimmunogical composition, including a vaccine, is effective against asingle multiple-banded antigen. The immunological compositions mayrecognize multiple serotypes of a biovar, in some cases. In specificcases, the immunological compositions recognize an antigen that isconserved between biovars.

Multiple banded antigen (MBA) is the predominant antigen recognizedduring infection with Ureaplasma and plays a role in virulence (Watson HL, et al Infect Immun 1990). It is species specific and contains crossreactive epitopes. The 5′ end of the MBA gene is relatively conservedbut contains some biovar and serovar specificity. The MBA contains asignal peptide and acylation site in the N-terminal region, while the Cterminal region is composed of multiple six-amino-acid (encoded by 18nucleotides) tandem repeats, which contain serovar-specific epitopes.Alteration of the copy number of the repeating units results in MBA sizevariation (Zheng X, et al. Ann NY Acad Sci 1994). In contrast to therepeat region, the 5′ region is conserved among serovar variants (Teng LJ, et al. J Clin Microbiol 1994). Although serovar specificity isdetermined by the composition of the C-terminal region of MBAs, there issome heterogeneity detected in the sequence of the 5′ region of the MBAgene of the different serovars which allows the 14 serovars to bedivided into several subgroups. Thus, in specific embodiments of theinvention, the compositions are focused on the more conserved regions ofthe MBA so that any vaccine, antigen, or antibody would be applicable toall or most serotypes, biovars, and even species. An exemplary sequencefor the conserved sequence is below:

Serotype 6 MAB cDNA sequence (AF056984) (SEQ ID NO: 4):   1GTATTTGCAA TCTTTATATG TTTTCGTTAA AATTAAAAAT TAATTACTAT AAAAATTATG  61TAAGATTAAT AAATCTTAGT GTTCATATTT TTTACTAGTA TTAAATTAAA AACAATAAAA 121TGACATATTT TTTATATTAG GAGAACCATA AATGAAATTA TTAAAAAATA AAAAATTCTG 181AGCTATGACA TTAGGAGTTA CCTTAGTTGG AGCTGGAATA GTTGCTATAG CGGCTTCATG 241TTCTAATTCA ACTGTTAAAT CTAAGTTAAG TAGCCAATTT GTTAAATCAA CAGATGATAA 301AAGTTTTTAT GCAGTTTACG AAATTGAAAA CTTTAAAGAT CTAAGTGATA ATGATAAAAA 361ATCATTAAAT GACATTGAAT TTAATGCTGC ACTTACATCA GTTGAAAACA AAACAGAAAA 421TCTAGTTACA AAAGGTCATT TGGTTGGTGA AAAAATTTAC GTTAAATTAC CTCGTGAACC 481AAAACCTAAT GAACAATTAA CTATTATTAA TAAAAGTGGA TTAATCAAGA CTTCAGGTTT 541GTTAATACCT AATAATTTGA ATTATCAAAC AGAAAAAGTG AACTTTGAAA CAGCTCCGAA 601AACTCAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 661AGGTAAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 721AGGTAAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 781AGGTAAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 841AGGTAAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 901AGGTAAAGAA CCAGGTAAAG AACCAGGTAA AGAACCAGGT AAAGAACCAG GTAAAGAACC 961AGGTAAAGAA

In some cases of the invention, an immunological compositionsimmunologically reacts with a multiple-banded antigen of Ureaplasma froma patient, for example, although the immunological composition itselfmay have been raised against an antigen having a slight modificationfrom the naturally occurring corresponding antigen. For example, anantibody may recognize the naturally occurring antigen, such as from apatient, although the antibody may have been raised against a peptide orpolypeptide sequence having less than 100% identity to the naturallyoccurring antigen. In specific embodiments, the antibody was raisedagainst peptide or polypeptide sequence having 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to the sequence in the naturally occurring antigen. In somecases, the antibody was raised against peptide or polypeptide having 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid differences compared tothe sequence of the naturally occurring antigen, yet the antibody stillrecognizes the sequence in the naturally occurring antigen.

III. Exemplary Vaccines of the Invention

In some embodiments of the invention, there are vaccines directedagainst one or more Ureaplasma antigens. The antigens may be themultiple-banded antigen or it may be another antigen.

In some embodiments of the invention, there are several othernon-Multiple Banded Antigen (MBA) antigens and they or their respectiveDNA could be targets for a vaccine or their antibody products forsimilar prevention and treatment strategies and possible diagnostictargets including: 1) the enzyme Urease that is necessary for theorganism's survival. There are several Ureases (A-G) known at this time(UU428, UU429, UU430, UU431, UU432, UU433, UU434); 2) Adjacent to theMBA gene (UU375) is gene UU376, which is a Ureaplasma-specific conservedhypothetical gene and another potential target; 3) There appears to bevirulence genes (hemolysin) including hlyC (UU072), hlyA (UU436) thatare useful targets, in some aspects; 4) MBA N-terminal paralogs (UU172,UU189, UU483, UU487, UU526). Phase variation of the multiple-bandedantigen (MBA) with its counterpart, the UU376 protein, results in DNAinversion at specific inverted repeats. These recombination events aredynamic and can lead to a broad spectrum of antigenic variation by whichthe organism could evade host immune responses; thus in specificembodiments these are targeted; 5) There are several other genes to alsoconsider including the following, for example: a) 16S rRNA genes, b) thegenes adjoining the urease genes including the area upstream of theUrease A gene, the Urease A-Urease B spacer, the Urease B-Urease Cspacer, c) the 16s-23S rRNA intergenic spacer region, and/or d) ureatransporter.

DNA Vaccine: In specific embodiments of the invention, the vaccines arecomposed of a piece of the pathogen's DNA (plasmid, for example)genetically engineered to produce at least one, two, or more specificproteins (antigens) from a pathogen. The plasmid DNA (pDNA) is injectedinto the cells of the body, where the host cells read the pDNA andproduces its antigens. These antigens are recognized as foreign whenproduced and displayed by the host cells, and the host immune systemtriggers a range of immune responses. (Alarcon et al., 1999; Robinsonand Pertmer, 2000)

Thus far, several DNA vaccines have been developed and many more areunder consideration. (Kutzler and Weiner, 2008) Specifically, positiveresults are seen for a bird flu DNA vaccine (Cinatl et al., 2007).Veterinary DNA vaccines have been approved to: 1) protect horses fromWest Nile virus (Fort Dodge Animal Health Announces Approval of WestNile Virus DNA Vaccine for Horses, P R Neswire 2005 Jul. 18); 2) protectsalmon from Infectious hematopoietic necrosis virus; 3) protect pigletsfrom perinatal mortality and morbidity due to weaning; 4) treats dogswith aggressive melanoma. A preliminary study for a DNA vaccine againstmultiple sclerosis was reported as being effective (Stuve et al., 2007).

There are several advantages and disadvantages for DNA vaccines.(Alarcon et al., 1999; Kutzler and Weiner, 2008; Robisnson and Pertmer,2000; Sedegah et al., 1994) The advantages include the following:subunit vaccination without risk for infection, antigen presentation byboth MHC class I and II molecules, ability to polarize T-cell helptoward type 1 or 2, immune response focused only on antigen(s) ofinterest, ease of development and production, stability of vaccine forstorage and shipping, cost-effectiveness, eliminates need for peptidesynthesis, expression, and purification of recombinant proteins and theuse of toxic adjuvants, long term persistence of immunogen, in vivoexpression ensures protein more closely resembles normal eukaryoticstructure, with accompanying post-translational modifications.

DNA Vaccine Development and Design: There are several methods tooptimize DNA vaccine development. 1) DNA vaccines appear to obtain thebest immune response when highly active expression vectors are used.Thus, a strong viral promoter to drive the in vivo transcription andtranslation of the DNA or complimentary DNA of interest is useful. (Moret al., 1995) In some embodiments the cytomegalovirus early promoter(CMV) is employed because it had higher expression rates than the SV40promoter or Rous Sarcoma Virus promoter. 2) In some embodiments there isincluded Mason-Pfizer monkey virus with rev (MPV)-CTE+rev increasesenvelope expression and is more immunogenic. (Muthumani et al., 2003)One can add the MPV-CTE+rev to a vaccine and attempt to increaseenvelope expression and immunogenicity. 3) An Intron A may sometimes beincluded in the plasmid vector to improve mRNA stability and thusincrease protein expression. (Leitner et al., 1997) pVAX1 by Invitrogenmay be included in the plasmid at this location, in some embodiments. Anewer more effective vector (e.g. pVAX200-DEST by Invitrogen) may beemployed. 4) Plasmids should also include a strongpolyadenylation/transcriptional termination signal such as a bovinegrowth hormone (BGH). (Alarcon et al., 1999; Bohm et al., 1996; Robinsonand Pertmer, 2000) The inventors have already done this with the pVAX1vector and BGH is also present on the pVAX200-DEST vector. 5) Vectorsthat express more than one immunogen may also enhance a vaccine'sefficacy and impact and be employed. (Carey et al., 1991) In some cases,there is expression of more than one immunogen in the plasmid to enhancethe vaccines efficacy and impact. Specifically, one can express themultiple binding proteins (MBP) from several serotypes and strains ofUreaplasma. 6) To optimize vector design for maximal protein expression,one can use a codon of pathogenic mRNA for eukaryotic cells. (Lewis andBabiuk, 1999) Pathogens often have different AT contents different thanthe species being immunized, so altering the gene sequence of theimmunogen to reflect codons more commonly used in the target species mayimprove its expression, in certain cases of the invention. (3) Inspecific embodiments, ANNATPG (SEQ ID NO:1) is employed in front of thestart code. The adenovirus tripartite leader (TPL) may be utilized incertain cases. (Kutzler and Weiner, 2008) Further enhancer sequences maybe utilized for the vaccine. In specific embodiments, humangranulocytemacrophage colony-stimulating factor (hGM-CSF) is used in thecurrent vaccine as an immune enhancer. (Kutzler and Weiner, 2008) 7)Immunogens can be targeted to various cellular compartments in order toimprove antibody or cytotoxic T-cell responses. Plasma membrane-boundantigens are more effective at inducing antibody responses thancytosolic antigens (Robinson and Pertmer, 2000), in certain cases, andsuch may be used in the vaccine. 8) Cytotoxic T-cell responses can beimproved by targeting antigens for cytoplasmic degradation andsubsequent entry into MHC class I pathway (Robinson and Pertmer, 2000)by the addition of N-terminal ubiquitin signals (Rodriguez et al., 1997)to the stop code, and in certain embodiments such is used in theinvention. 9) Conformation of the protein can also have an effect onantibody responses, with ordered structures being more effective thanunordered structures, and it may be used in the invention. (Wunderlichet al., 2000) 10) Strings of MHC class I epitopes from differentpathogens or oligonucleotides (e.g. CpG motif) are able to raisecytotoxic T-cell responses to a number of pathogens, especially if a THepitope is also included (Robinson and Pertmer, 2000), and such may beemployed in certain embodiments.

DNA Vaccine Delivery: DNA vaccines have been introduced into animaltissues by several different methods. (Weiner and Kennedy, 1999) Thesedelivery methods include the following: 1) Injection via a hypodermicneedle of an aqueous solution of DNA in saline by intramuscular (IM),intradermal (ID), intravenous (IV), subcutaneous (SC), orintraperitoneal (IP) route. The latter three have had variable successand all require large amounts of DNA (100-200 ug). Although these arenot specialized delivery mechanisms, they are simple, lead to permanentor semi-permanent expression, lead to pDNA spread rapidly throughout thebody. However, they are inefficient in their uptake, require relativelylarge amounts of DNA, and the Th1 response may not be the responserequired. Although several methods can be used to increase delivery tothe cell including electroporation (Widera et al., 2000), damagingmuscle fibers with myotoxins (e.g. bupivacaine) or hypertonic solutions(e.g. saline or sucrose), or more damaging injection (needle type,needle alignment, speed of injection, volume of injection) (2). Lack ofpractical application of these methods or their side effects potentiallyoutweigh their benefits and at least in some cases they are notutilized. 2) Gene Gun delivery of a DNA coated gold or tungsten beadsvia epidermal delivery (ED) through the skin or outer membrane (vaginalmucosa), or surgically exposed muscle or other organs. This methodallows the DNA to be bombarded directly into cells utilizing compressedhelium as an accelerant, and requires a small amount of DNA (as littleas 16 ng). However, the disadvantage is that the Th2 response may not berequired and inert particles are required as carriers. 3) Pneumatic(Jet) injection of an aqueous solution of DNA by ED. The advantage isthat no particles are required, DNA can be delivered to cells mm to cmbelow the skin or tissue surface. The disadvantage is there issignificant shearing of DNA after high-pressure expulsion, a 10-foldlower expression and lower immune response has been reported, and itrequires large amounts of DNA (up to 300 ug). 4) Liposome mediateddelivery of several of the above-mentioned systems but particularly IM,IV, IP, and Oral or Mucosal (Nasal, Vaginal) has several advantages. Itcan increase the immune response substantially, increase thetransfection of pDNA, and mucosally delivered liposomal-DNA complexescan result in expression at distal mucosa and the generation of IgAantibodies. The potential disadvantages are the variability of theresponse and thus ineffectiveness, and the possibility of toxicitysecondary to the enhanced immune response. For certain cases theinventors employed the IP route with an aqueous solution to allow themost generalized distribution of the pDNA while minimizing thecomplexities of delivering a vaccine IV or using a more expensivedelivery system (Gene Gun) or complex media (Liposome). Regardless ofthese methods, several factors can influence the immune responsesrelated to injections including age and sex and may be considered incertain embodiments of the invention.

IV. General Vaccine Embodiments

For an antigenic composition to be useful as a vaccine, an antigeniccomposition must induce an immune response to the antigen in a cell,tissue or animal (e.g., a mammal, including a human). As used herein, an“antigenic composition” may comprise an antigen (e.g., a peptide orpolypeptide), a nucleic acid encoding an antigen (e.g., an antigenexpression vector), or a cell expressing or presenting an antigen. Inparticular embodiments the antigenic composition comprises or encodesall or part of a peptide or polypeptide sequence, or an immunologicallyfunctional equivalent thereof. In other embodiments, the antigeniccomposition is in a mixture that comprises an additionalimmunostimulatory agent or nucleic acids encoding such an agent.Immunostimulatory agents include but are not limited to an additionalantigen, an immunomodulator, an antigen presenting cell or an adjuvant.In other embodiments, one or more of the additional agent(s) iscovalently bonded to the antigen or an immunostimulatory agent, in anycombination. In certain embodiments, the antigenic composition isconjugated to or comprises an HLA anchor motif amino acids.

In certain embodiments, an antigenic composition or immunologicallyfunctional equivalent, may be used as an effective vaccine in inducingan anti Ureaplasma humoral and/or cell mediated immune response in ananimal. The present invention contemplates one or more antigeniccompositions or vaccines for use in both active and passive immunizationembodiments.

A vaccine of the present invention may vary in its composition ofproteinaceous, nucleic acid and/or cellular components. In anon-limiting example, a nucleic acid encoding an antigen might also beformulated with a proteinaceous adjuvant. Of course, it will beunderstood that various compositions described herein may furthercomprise additional components. For example, one or more vaccinecomponents may be comprised in a lipid or liposome. In anothernon-limiting example, a vaccine may comprise one or more adjuvants. Avaccine of the present invention, and its various components, may beprepared and/or administered by any method disclosed herein or as wouldbe known to one of ordinary skill in the art, in light of the presentdisclosure.

A. Proteinaceous Antigens

It is understood that an antigenic composition of the present inventionmay be made by a method that is well known in the art, including but notlimited to chemical synthesis by solid phase synthesis and purificationaway from the other products of the chemical reactions by HPLC, orproduction by the expression of a nucleic acid sequence (e.g., a DNAsequence) encoding a peptide or polypeptide comprising an antigen of thepresent invention in an in vitro translation system or in a living cell.Preferably the antigenic composition is isolated and extensivelydialyzed to remove one or more undesired small molecular weightmolecules and/or lyophilized for more ready formulation into a desiredvehicle. It is further understood that additional amino acids,mutations, chemical modification and such like, if any, that are made ina vaccine component will preferably not substantially interfere with theantibody recognition of the epitopic sequence.

A peptide or polypeptide corresponding to one or more antigenicdeterminants of the Ureaplasma of the present invention should generallybe at least five or six amino acid residues in length, and may containup to about 10, about 15, about 20, about 25 about 30, about 35, about40, about 45 or about 50 or more residues or so. A peptide sequence maybe synthesized by methods known to those of ordinary skill in the art,such as, for example, peptide synthesis using automated peptidesynthesis machines, such as those available from Applied Biosystems(Foster City, Calif.).

Longer peptides or polypeptides also may be prepared, e.g., byrecombinant means. In certain embodiments, a nucleic acid encoding anantigenic composition and/or a component described herein may be used,for example, to produce an antigenic composition in vitro or in vivo forthe various compositions and methods of the present invention. Forexample, in certain embodiments, a nucleic acid encoding an antigen iscomprised in, for example, a vector in a recombinant cell. The nucleicacid may be expressed to produce a peptide or polypeptide comprising anantigenic sequence. The peptide or polypeptide may be secreted from thecell, or comprised as part of or within the cell.

B. Proteinaceous Antigens

In certain embodiments, an immune response may be promoted bytransfecting or inoculating an animal with a nucleic acid encoding anantigen. One or more cells comprised within a target animal thatexpresses the sequences encoded by the nucleic acid after administrationof the nucleic acid to the animal. Thus, the vaccine may comprise“genetic vaccine” useful for immunization protocols. A vaccine may alsobe in the form, for example, of a nucleic acid (e.g., a cDNA or an RNA)encoding all or part of the peptide or polypeptide sequence of anantigen. Expression in vivo by the nucleic acid may be, for example, bya plasmid type vector, a viral vector, or a viral/plasmid constructvector.

In preferred aspects, the nucleic acid comprises a coding region thatencodes part of the sequences from Ureaplasma, or an immunologicallyfunctional equivalent thereof. Of course, the nucleic acid may compriseand/or encode additional sequences, including but not limited to thosecomprising one or more immunomodulators or adjuvants. The nucleotide andprotein, polypeptide and peptide encoding sequences for various geneshave been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. One such databaseis the National Center for Biotechnology Information's GenBank® andGenPept databases. The coding regions for these known genes may beamplified, combined with the sequences encompassed in the invention(e.g., ligated) and/or expressed using the techniques disclosed hereinor by any technique that would be know to those of ordinary skill in theart (e.g., Sambrook et al., 1987). Though a nucleic acid may beexpressed in an in vitro expression system, in preferred embodiments thenucleic acid comprises a vector for in vivo replication and/orexpression.

C. Cellular Vaccine Antigens

In another embodiment, a cell expressing the antigen may comprise thevaccine. The cell may be isolated from a culture, tissue, organ ororganism and administered to an animal as a cellular vaccine. Thus, thepresent invention contemplates a “cellular vaccine.” The cell may betransfected with a nucleic acid encoding an antigen to enhance itsexpression of the antigen. Of course, the cell may also express one ormore additional vaccine components, such as immunomodulators oradjuvants. A vaccine may comprise all or part of the cell.

In particular embodiments, it is contemplated that nucleic acidsencoding antigens of the present invention may be transfected intoplants, particularly edible plants, and all or part of the plantmaterial used to prepare a vaccine, such as for example, an oralvaccine. Such methods are described in U.S. Pat. Nos. 5,484,719,5,612,487, 5,914,123, 5,977,438 and 6,034,298, each incorporated hereinby reference.

D. Immunologically Functional Equivalents

As modifications and changes may be made in the structure of anantigenic composition of the present invention, and still obtainmolecules having like or otherwise desirable characteristics, suchimmunologically functional equivalents are also encompassed within thepresent invention.

For example, certain amino acids may be substituted for other aminoacids in a peptide, polypeptide or protein structure without appreciableloss of interactive binding capacity with structures such as, forexample, antigen binding regions of antibodies, binding sites onsubstrate molecules or receptors, DNA binding sites, or such like. Sinceit is the interactive capacity and nature of a peptide, polypeptide orprotein that defines its biological (e.g., immunological) functionalactivity, certain amino acid sequence substitutions can be made in aamino acid sequence (or, of course, its underlying DNA coding sequence)and nevertheless obtain a peptide or polypeptide with like (agonistic)properties. It is thus contemplated by the inventors that variouschanges may be made in the sequence of an antigenic composition such as,for example a peptide or polypeptide, or underlying DNA, withoutappreciable loss of biological utility or activity.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of the antigeniccomposition comprises amino molecules that are sequential, without anynon-amino molecule interrupting the sequence of amino molecule residues.In other embodiments, the sequence may comprise one or more non-aminomolecule moieties. In particular embodiments, the sequence of residuesof the antigenic composition may be interrupted by one or more non-aminomolecule moieties.

Accordingly, antigenic composition, particularly an immunologicallyfunctional equivalent of the sequences disclosed herein, may encompassan amino molecule sequence comprising at least one of the 20 commonamino acids in naturally synthesized proteins, or at least one modifiedor unusual amino acid.

In term of immunologically functional equivalent, it is well understoodby the skilled artisan that, inherent in the definition is the conceptthat there is a limit to the number of changes that may be made within adefined portion of the molecule and still result in a molecule with anacceptable level of equivalent immunological activity. Animmunologically functional equivalent peptide or polypeptide are thusdefined herein as those peptide(s) or polypeptide(s) in which certain,not most or all, of the amino acid(s) may be substituted.

In particular, where a shorter length peptide is concerned, it iscontemplated that fewer amino acid substitutions should be made withinthe given peptide. A longer polypeptide may have an intermediate numberof changes. The full length protein will have the most tolerance for alarger number of changes. Of course, a plurality of distinctpolypeptides/peptides with different substitutions may easily be madeand used in accordance with the invention.

It also is well understood that where certain residues are shown to beparticularly important to the immunological or structural properties ofa protein or peptide, e.g., residues in binding regions or active sites,such residues may not generally be exchanged. This is an importantconsideration in the present invention, where changes in the antigenicsite should be carefully considered and subsequently tested to ensuremaintenance of immunological function (e.g., antigenicity), wheremaintenance of immunological function is desired. In this manner,functional equivalents are defined herein as those peptides orpolypeptides which maintain a substantial amount of their nativeimmunological activity.

Amino acid substitutions are generally based on the relative similarityof the amino acid side chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as immunologically functionalequivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan(0.9); tyro sine (1.3); proline (1.6); histidine (3.2); glutamate (3.5);glutamine (3.5); aspartate (3.5); asparagine (3.5); lysine (3.9); andarginine (4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein, polypeptide or peptide isgenerally understood in the art (Kyte & Doolittle, 1982, incorporatedherein by reference). It is known that certain amino acids may besubstituted for other amino acids having a similar hydropathic index orscore and still retain a similar biological activity. In making changesbased upon the hydropathic index, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those which are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the immunological functional equivalent polypeptideor peptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a immunological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (0.4);proline (0.5±1); alanine (0.5); histidine (0.5); cysteine (1.0);methionine (1.3); valine (1.5); leucine (1.8); isoleucine (1.8);tyrosine (2.3); phenylalanine (2.5); tryptophan (3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

Numerous scientific publications have also been devoted to theprediction of secondary structure, and to the identification of anepitope, from analyses of an amino acid sequence (Chou & Fasman,1974a,b; 1978a,b, 1979). Any of these may be used, if desired, tosupplement the teachings of U.S. Pat. No. 4,554,101.

Moreover, computer programs are currently available to assist withpredicting an antigenic portion and an epitopic core region of one ormore proteins, polypeptides or peptides. Examples include those programsbased upon the Jameson Wolf analysis (Jameson & Wolf, 1988; Wolf et al.,1988), the program PepPlot® (Brutlag et al., 1990; Weinberger et al.,1985), and other new programs for protein tertiary structure prediction(Fetrow & Bryant, 1993). Another commercially available software programcapable of carrying out such analyses is MacVector (IBI, New Haven,Conn.).

In further embodiments, major antigenic determinants of a peptide orpolypeptide may be identified by an empirical approach in which portionsof a nucleic acid encoding a peptide or polypeptide are expressed in arecombinant host, and the resulting peptide(s) or polypeptide(s) testedfor their ability to elicit an immune response. For example, PC™ can beused to prepare a range of peptides or polypeptides lacking successivelylonger fragments of the C terminus of the amino acid sequence. Theimmunoactivity of each of these peptides or polypeptides is determinedto identify those fragments or domains that are immunodominant. Furtherstudies in which only a small number of amino acids are removed at eachiteration then allows the location of the antigenic determinant(s) ofthe peptide or polypeptide to be more precisely determined.

Another method for determining a major antigenic determinant of apeptide or polypeptide is the SPOTs system (Genosys Biotechnologies,Inc., The Woodlands, Tex.). In this method, overlapping peptides aresynthesized on a cellulose membrane, which following synthesis anddeprotection, is screened using a polyclonal or monoclonal antibody. Anantigenic determinant of the peptides or polypeptides which areinitially identified can be further localized by performing subsequentsyntheses of smaller peptides with larger overlaps, and by eventuallyreplacing individual amino acids at each position along theimmunoreactive sequence.

Once one or more such analyses are completed, an antigenic composition,such as for example a peptide or a polypeptide is prepared that containat least the essential features of one or more antigenic determinants.An antigenic composition is then employed in the generation of antiseraagainst the composition, and preferably the antigenic determinant(s).

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid. Nucleic acids encodingthese antigenic compositions also can be constructed and inserted intoone or more expression vectors by standard methods (Sambrook et al.,1987), for example, using PCR cloning methodology.

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide or polypeptidestructure or to interact specifically with, for example, an antibody.Such compounds, which may be termed peptidomimetics, may be used in thesame manner as a peptide or polypeptide of the invention and hence arealso immunologically functional equivalents.

Certain mimetics that mimic elements of protein secondary structure aredescribed in Johnson et al. (1993). The underlying rationale behind theuse of peptide mimetics is that the peptide backbone of proteins existschiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is thus designed to permit molecularinteractions similar to the natural molecule.

E. Antigen Mutagenesis

In particular embodiments, an antigenic composition is mutated forpurposes such as, for example, enhancing its immunogenicity or producingor identifying a immunologically functional equivalent sequence. Methodsof mutagenesis are well known to those of skill in the art (Sambrook etal., 1987).

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template dependent processes and vector mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In a preferred embodiment, site directed mutagenesis is used. Sitespecific mutagenesis is a technique useful in the preparation of anantigenic composition (e.g., a composition comprising peptide orpolypeptide, or immunologically functional equivalent protein,polypeptide or peptide), through specific mutagenesis of the underlyingDNA. In general, the technique of site specific mutagenesis is wellknown in the art. The technique further provides a ready ability toprepare and test sequence variants, incorporating one or more of theforegoing considerations, by introducing one or more nucleotide sequencechanges into the DNA. Site specific mutagenesis allows the production ofa mutant through the use of specific oligonucleotide sequence(s) whichencode the DNA sequence of the desired mutation, as well as a sufficientnumber of adjacent nucleotides, to provide a primer sequence ofsufficient size and sequence complexity to form a stable duplex on bothsides of the position being mutated. Typically, a primer of about 17 toabout 75 nucleotides in length is preferred, with about 10 to about 25or more residues on both sides of the position being altered, whileprimers of about 17 to about 25 nucleotides in length being morepreferred, with about 5 to 10 residues on both sides of the positionbeing altered.

In general, site directed mutagenesis is performed by first obtaining asingle stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. As will be appreciated by one of ordinary skill in theart, the technique typically employs a bacteriophage vector that existsin both a single stranded and double stranded form. Typical vectorsuseful in site directed mutagenesis include vectors such as the M13phage. These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

This mutagenic primer is then annealed with the single stranded DNApreparation, and subjected to DNA polymerizing enzymes such as, forexample, E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected that include recombinant vectors bearing the mutatedsequence arrangement.

Alternatively, a pair of primers may be annealed to two separate strandsof a double stranded vector to simultaneously synthesize bothcorresponding complementary strands with the desired mutation(s) in aPCR™ reaction. A genetic selection scheme to enrich for clonesincorporating the mutagenic oligonucleotide has been devised (Kunkel etal., 1987). Alternatively, the use of PCR with commercially availablethermostable enzymes such as Taq polymerase may be used to incorporate amutagenic oligonucleotide primer into an amplified DNA fragment that canthen be cloned into an appropriate cloning or expression vector (Tomicet al., 1990; Upender et al., 1995). A PCR employing a thermostableligase in addition to a thermostable polymerase also may be used toincorporate a phosphorylated mutagenic oligonucleotide into an amplifiedDNA fragment that may then be cloned into an appropriate cloning orexpression vector (Michael 1994).

The preparation of sequence variants of the selected gene using sitedirected mutagenesis is provided as a means of producing potentiallyuseful species and is not meant to be limiting, as there are other waysin which sequence variants of genes may be obtained. For example,recombinant vectors encoding the desired gene may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

Additionally, one particularly useful mutagenesis technique is alaninescanning mutagenesis in which a number of residues are substitutedindividually with the amino acid alanine so that the effects of losingside chain interactions can be determined, while minimizing the risk oflarge scale perturbations in protein conformation (Cunningham et al.,1989).

F. Vectors

In some embodiments of the invention, an immunological compositioncomprising a nucleic acid vector is employed.

In order to effect replication, expression or mutagenesis of a nucleicacid, the nucleic acid may be delivered (“transfected”) into a cell. Thetransfection of cells may be used, in certain embodiments, torecombinately produce one or more vaccine components for subsequentpurification and preparation into a pharmaceutical vaccine. In otherembodiments, the nucleic acid may be comprised as a genetic vaccine thatis administered to an animal. In other embodiments, the nucleic acid istransfected into a cell and the cell administered to an animal as acellular vaccine component. The nucleic acid may consist only of nakedrecombinant DNA, or may comprise, for example, additional materials toprotect the nucleic acid and/or aid its targeting to specific celltypes.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell.

The nucleic acid encoding the antigenic composition or other vaccinecomponent may be stably integrated into the genome of the cell, or maybe stably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. Vectors and expression vectors may containnucleic acid sequences that serve other functions as well and aredescribed infra. How the expression construct is delivered to a cell andwhere in the cell the nucleic acid remains is dependent on the type ofexpression construct employed.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30 110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include the βlactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base, EPDB) could also be used to driveexpression. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Nonlimiting examples of such regions include the human LIMK2 gene(Nomoto et al. 1999), the somatostatin receptor 2 gene (Kraus et al.,1998), murine epididymal retinoic acid-binding gene (Lareyre et al.,1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen(Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee, et al., 1997),insulin-like growth factor II (Wu et al., 1997), and human plateletendothelial cell adhesion molecule-1 (Almendro et al., 1996).

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

5. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

6. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

7. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

8. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

9. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector ordinarilycarries a replication site, as well as marking sequences which arecapable of providing phenotypic selection in transformed cells. In anon-limiting example, E. coli is often transformed using derivatives ofpBR322, a plasmid derived from an E. coli species. pBR322 contains genesfor ampicillin and tetracycline resistance and thus provides easy meansfor identifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, forexample, promoters which can be used by the microbial organism forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM 11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as, for example,E. coli LE392.

Further useful plasmid vectors include pIN vectors (Inouye et al.,1985); and pGEX vectors, for use in generating glutathione S transferase(GST) soluble fusion proteins for later purification and separation orcleavage. Other suitable fusion proteins are those with galactosidase,ubiquitin, and the like.

Bacterial host cells, for example, E. coli, comprising the expressionvector, are grown in any of a number of suitable media, for example, LB.The expression of the recombinant protein in certain vectors may beinduced, as would be understood by those of skill in the art, bycontacting a host cell with an agent specific for certain promoters,e.g., by adding IPTG to the media or by switching incubation to a highertemperature. After culturing the bacteria for a further period,generally of between 2 and 24 h, the cells are collected bycentrifugation and washed to remove residual media.

10. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Vaccine components of the present invention may be aviral vector that encode one or more antigenic compositions or othercomponents such as, for example, an immunomodulator or adjuvant.Non-limiting examples of virus vectors that may be used to deliver anucleic acid of the present invention are described below.

11. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a 36 kb, linear, double stranded DNAvirus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus and Horwitz, 1992).

12. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno associated virus(AAV) is an attractive vector system for use in the [INVENTION] vaccinesof the present invention as it has a high frequency of integration andit can infect nondividing cells, thus making it useful for delivery ofgenes into mammalian cells, for example, in tissue culture (Muzyczka,1992) or in vivo. AAV has a broad host range for infectivity (Tratschinet al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlinet al., 1988). Details concerning the generation and use of rAAV vectorsare described in U.S. Pat. Nos. 5,139,941 and 4,797,368, eachincorporated herein by reference.

13. Retroviral Vectors

Retroviruses have promise as antigen delivery vectors in vaccines due totheir ability to integrate their genes into the host genome,transferring a large amount of foreign genetic material, infecting abroad spectrum of species and cell types and of being packaged inspecial cell lines (Miller, 1992).

In order to construct a vaccine retroviral vector, a nucleic acid (e.g.,one encoding an antigen of interest) is inserted into the viral genomein the place of certain viral sequences to produce a virus that isreplication defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression require the division of host cells (Paskind et al., 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomeret al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 andthe Simian Immunodeficiency Virus: SIV. Lentiviral vectors have beengenerated by multiply attenuating the HIV virulence genes, for example,the genes env, vif, vpr, vpu and nef are deleted making the vectorbiologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

14. Other Viral Vectors

Other viral vectors may be employed as vaccine constructs in the presentinvention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and herpes simplex virus may be employed.They offer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988; Horwich et al., 1990).

G. Vaccine Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was recentlydeveloped based on the chemical modification of a retrovirus by thechemical addition of lactose residues to the viral envelope. Thismodification can permit the specific infection of hepatocytes viasialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989). Thus, it is contemplated that antibodies,specific binding ligands and/or other targeting moieties may be used tospecifically transfect APC types.

H. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harlan and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991) and receptor-mediatedtransfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); by Agrobacteriummediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055, eachincorporated herein by reference); or by PEG mediated transformation ofprotoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and4,952,500, each incorporated herein by reference); bydesiccation/inhibition mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

1. Injection

In certain embodiments, a nucleic acid may be delivered to an organelle,a cell, a tissue or an organism via one or more injections (i.e., aneedle injection). Methods of injection of nucleic acids are describedherein, and are well known to those of ordinary skill in the art.Further embodiments of the present invention include the introduction ofa nucleic acid by direct microinjection to a cell. Direct microinjectionhas been used to introduce nucleic acid constructs into Xenopus oocytes(Harland and Weintraub, 1985). The amount of composition used may varyupon the nature of the antigen as well as the organelle, cell, tissue ororganism used

2. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into an organelle, a cell, a tissue or an organism viaelectroporation. Electroporation involves the exposure of a suspensionof cells and DNA to a high voltage electric discharge. In some variantsof this method, certain cell wall degrading enzymes, such as pectindegrading enzymes, are employed to render the target recipient cellsmore susceptible to transformation by electroporation than untreatedcells (U.S. Pat. No. 5,384,253, incorporated herein by reference).Alternatively, recipient cells can be made more susceptible totransformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre B lymphocytes have been transfected with humankappa immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

3. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV 1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

4. DEAE Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

5. Sonication Loading

Additional embodiments of the present invention include the introductionof a nucleic acid by direct sonic loading. LTK fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

I. Liposome Mediated Transfection

In a further embodiment of the invention, one or more vaccine componentsor nucleic acids may be entrapped in a lipid complex such as, forexample, a liposome. Liposomes are vesicular structures characterized bya phospholipid bilayer membrane and an inner aqueous medium.Multilamellar liposomes have multiple lipid layers separated by aqueousmedium. They form spontaneously when phospholipids are suspended in anexcess of aqueous solution. The lipid components undergo selfrearrangement before the formation of closed structures and entrap waterand dissolved solutes between the lipid bilayers (Ghosh and Bachhawat,1991). Also contemplated is an nucleic acid complexed with Lipofectamine(Gibco BRL) or Superfect (Qiagen).

Liposome mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry of liposomeencapsulated DNA (Kaneda et al., 1989). In other embodiments, a liposomemay be complexed or employed in conjunction with nuclear non histonechromosomal proteins (HMG 1) (Kato et al., 1991). In yet furtherembodiments, a liposome may be complexed or employed in conjunction withboth HVJ and HMG 1. In other embodiments, a delivery vehicle maycomprise a ligand and a liposome.

J. Receptor Mediated Transfection

One or more vaccine components or nucleic acids, may be employed todelivered using a receptor mediated delivery vehicle. These takeadvantage of the selective uptake of macromolecules by receptor mediatedendocytosis that will be occurring in the target cells. In view of thecell type specific distribution of various receptors, this deliverymethod adds another degree of specificity to the present invention.Specific delivery in the context of another mammalian cell type has beendescribed (Wu and Wu, 1993, incorporated herein by reference).

Certain receptor mediated gene targeting vehicles comprise a cellreceptor specific ligand and a nucleic acid binding agent. Otherscomprise a cell receptor specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell specific binding. For example, lactosyl ceramide, a galactoseterminal asialganglioside, have been incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes(Nicolau et al., 1987). It is contemplated that the tissue specifictransforming constructs of the present invention can be specificallydelivered into a target cell in a similar manner.

K. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No.5,610,042; and PCT Application WO 94/09699; each of which isincorporated herein by reference). This method depends on the ability toaccelerate DNA coated microprojectiles to a high velocity allowing themto pierce cell membranes and enter cells without killing them (Klein etal., 1987). There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

In microprojectile bombardment, one or more particles may be coated withat least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into a cell(e.g., a plant cell) by acceleration is the Biolistics Particle DeliverySystem, which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or Nytex screen, onto afilter surface covered with cells, such as for example, a monocot plantcells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

L. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny. Asused herein, the terms “engineered” and “recombinant” cells or hostcells are intended to refer to a cell into which an exogenous nucleicacid sequence, such as, for example, a vector, has been introduced.Therefore, recombinant cells are distinguishable from naturallyoccurring cells which do not contain a recombinantly introduced nucleicacid.

In certain embodiments, it is contemplated that RNAs or proteinaceoussequences may be co expressed with other selected RNAs or proteinaceoussequences in the same host cell. Co expression may be achieved by cotransfecting the host cell with two or more distinct recombinantvectors. Alternatively, a single recombinant vector may be constructedto include multiple distinct coding regions for RNAs, which could thenbe expressed in host cells transfected with the single vector.

A tissue may comprise a host cell or cells to be transformed with acomposition of the invention. The tissue may be part or separated froman organism. In certain embodiments, a tissue may comprise, but is notlimited to, adipocytes, alveolar, ameloblasts, axon, basal cells, blood(e.g., lymphocytes), blood vessel, bone, bone marrow, brain, breast,cartilage, cervix, colon, cornea, embryonic, endometrium, endothelial,epithelial, esophagus, facia, fibroblast, follicular, ganglion cells,glial cells, goblet cells, kidney, liver, lung, lymph node, muscle,neuron, ovaries, pancreas, peripheral blood, prostate, skin, skin, smallintestine, spleen, stem cells, stomach, or testes.

In certain embodiments, the host cell or tissue may be comprised in atleast one organism. In certain embodiments, the organism may be, but isnot limited to, a prokayote (e.g., a eubacteria, an archaea) or aneukaryote, as would be understood by one of ordinary skill in the art.

Numerous cell lines and cultures are available for use as a host cell,and they can be obtained through the American Type Culture Collection(ATCC), which is an organization that serves as an archive for livingcultures and genetic materials. An appropriate host can be determined byone of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Cell typesavailable for vector replication and/or expression include, but are notlimited to, bacteria, such as E. coli (e.g., E. coli strain RR1, E. coliLE392, E. coli B, E. coli×1776 (ATCC No. 31537) as well as E. coli W3110(F, lambda, prototrophic, ATCC No. 273325), DH5α, JM109, and KC8,bacilli such as Bacillus subtilis; and other enterobacteriaceae such asSalmonella typhimurium, Serratia marcescens, various Pseudomonas specie,as well as a number of commercially available bacterial hosts such asSURE® Competent Cells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla).In certain embodiments, bacterial cells such as E. coli LE392 areparticularly contemplated as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, Cos,CHO, Saos, and PC12. Many host cells from various cell types andorganisms are available and would be known to one of skill in the art.Similarly, a viral vector may be used in conjunction with either aeukaryotic or prokaryotic host cell, particularly one that is permissivefor replication or expression of the vector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

M. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'s COMPLETECONTROL Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

It is contemplated that the proteins, polypeptides or peptides producedby the methods of the invention may be “overexpressed”, i.e., expressedin increased levels relative to its natural expression in cells. Suchoverexpression may be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and direct methodsare preferred, for example, those involving SDS/PAGE and proteinstaining or western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein, polypeptide or peptide incomparison to the level in natural cells is indicative ofoverexpression, as is a relative abundance of the specific protein,polypeptides or peptides in relation to the other proteins produced bythe host cell and, e.g., visible on a gel.

In some embodiments, the expressed proteinaceous sequence forms aninclusion body in the host cell, the host cells are lysed, for example,by disruption in a cell homogenizer, washed and/or centrifuged toseparate the dense inclusion bodies and cell membranes from the solublecell components. This centrifugation can be performed under conditionswhereby the dense inclusion bodies are selectively enriched byincorporation of sugars, such as sucrose, into the buffer andcentrifugation at a selective speed. Inclusion bodies may be solubilizedin solutions containing high concentrations of urea (e.g. 8M) orchaotropic agents such as guanidine hydrochloride in the presence ofreducing agents, such as beta-mercaptoethanol or DTT (dithiothreitol),and refolded into a more desirable conformation, as would be known toone of ordinary skill in the art.

N. Vaccine Component Purification

In any case, a vaccine component (e.g., an antigenic peptide orpolypeptide or nucleic acid encoding a proteinaceous composition) may beisolated and/or purified from the chemical synthesis reagents, cell orcellular components. In a method of producing the vaccine component,purification is accomplished by any appropriate technique that isdescribed herein or well known to those of skill in the art (e.g.,Sambrook et al., 1987). Although preferred for use in certainembodiments, there is no general requirement that an antigeniccomposition of the present invention or other vaccine component alwaysbe provided in their most purified state. Indeed, it is contemplatedthat less substantially purified vaccine component, which is nonethelessenriched in the desired compound, relative to the natural state, willhave utility in certain embodiments, such as, for example, totalrecovery of protein product, or in maintaining the activity of anexpressed protein. However, it is contemplate that inactive productsalso have utility in certain embodiments, such as, e.g., in determiningantigenicity via antibody generation.

The present invention also provides purified, and in preferredembodiments, substantially purified vaccines or vaccine components. Theterm “purified vaccine component” as used herein, is intended to referto at least one vaccine component (e.g., a proteinaceous composition,isolatable from cells), wherein the component is purified to any degreerelative to its naturally obtainable state, e.g., relative to its puritywithin a cellular extract or reagents of chemical synthesis. In certainaspects wherein the vaccine component is a proteinaceous composition, apurified vaccine component also refers to a wild type or mutant protein,polypeptide, or peptide free from the environment in which it naturallyoccurs.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific compound (e.g., a protein,polypeptide, or peptide) forms the major component of the composition,such as constituting about 50% of the compounds in the composition ormore. In preferred embodiments, a substantially purified vaccinecomponent will constitute more than about 60%, about 70%, about 80%,about 90%, about 95%, about 99% or even more of the compounds in thecomposition.

In certain embodiments, a vaccine component may be purified tohomogeneity. As applied to the present invention, “purified tohomogeneity,” means that the vaccine component has a level of puritywhere the compound is substantially free from other chemicals,biomolecules or cells. For example, a purified peptide, polypeptide orprotein will often be sufficiently free of other protein components sothat degradative sequencing may be performed successfully. Variousmethods for quantifying the degree of purification of a vaccinecomponent will be known to those of skill in the art in light of thepresent disclosure. These include, for example, determining the specificprotein activity of a fraction (e.g., antigenicity), or assessing thenumber of polypeptides within a fraction by gel electrophoresis.

Various techniques suitable for use in chemical, biomolecule orbiological purification, well known to those of skill in the art, may beapplicable to preparation of a vaccine component of the presentinvention. These include, for example, precipitation with ammoniumsulfate, PEG, antibodies and the like or by heat denaturation, followedby centrifugation; fractionation, chromatographic procedures, includingbut not limited to, partition chromatograph (e.g., paper chromatograph,thin-layer chromatograph (TLC), gas-liquid chromatography and gelchromatography) gas chromatography, high performance liquidchromatography, affinity chromatography, supercritical flowchromatography ion exchange, gel filtration, reverse phase,hydroxylapatite, lectin affinity; isoelectric focusing and gelelectrophoresis (see for example, Sambrook et al. 1989; and Freifelder,Physical Biochemistry, Second Edition, pages 238 246, incorporatedherein by reference).

Given many DNA and proteins are known (see for example, the NationalCenter for Biotechnology Information's GenBank® and GenPept databases),or may be identified and amplified using the methods described herein,any purification method for recombinately expressed nucleic acid orproteinaceous sequences known to those of skill in the art can now beemployed. In certain aspects, a nucleic acid may be purified onpolyacrylamide gels, and/or cesium chloride centrifugation gradients, orby any other means known to one of ordinary skill in the art (see forexample, Sambrook et al. 1989, incorporated herein by reference). Infurther aspects, a purification of a proteinaceous sequence may beconducted by recombinately expressing the sequence as a fusion protein.Such purification methods are routine in the art. This is exemplified bythe generation of an specific protein glutathione S transferase fusionprotein, expression in E. coli, and isolation to homogeneity usingaffinity chromatography on glutathione agarose or the generation of apolyhistidine tag on the N or C terminus of the protein, and subsequentpurification using Ni affinity chromatography. In particular aspects,cells or other components of the vaccine may be purified by flowcytometry. Flow cytometry involves the separation of cells or otherparticles in a liquid sample, and is well known in the art (see, forexample, U.S. Pat. Nos. 3,826,364, 4,284,412, 4,989,977, 4,498,766,5,478,722, 4,857,451, 4,774,189, 4,767,206, 4,714,682, 5,160,974 and4,661,913). Any of these techniques described herein, and combinationsof these and any other techniques known to skilled artisans, may be usedto purify and/or assay the purity of the various chemicals,proteinaceous compounds, nucleic acids, cellular materials and/or cellsthat may comprise a vaccine of the present invention. As is generallyknown in the art, it is believed that the order of conducting thevarious purification steps may be changed, or that certain steps may beomitted, and still result in a suitable method for the preparation of asubstantially purified antigen or other vaccine component.

O. Additional Vaccine Components

It is contemplated that an antigenic composition of the invention may becombined with one or more additional components to form a more effectivevaccine. Non-limiting examples of additional components include, forexample, one or more additional antigens, immunomodulators or adjuvantsto stimulate an immune response to an antigenic composition of thepresent invention and/or the additional component(s).

1. Immunomodulators

For example, it is contemplated that immunomodulators can be included inthe vaccine to augment a cell's or a patient's (e.g., an animal's)response. Immunomodulators can be included as purified proteins, nucleicacids encoding immunomodulators, and/or cells that expressimmunomodulators in the vaccine composition. The following sections listnon-limiting examples of immunomodulators that are of interest, and itis contemplated that various combinations of immunomodulators may beused in certain embodiments (e.g., a cytokine and a chemokine).

2. Cytokines

Interleukins, cytokines, nucleic acids encoding interleukins orcytokines, and/or cells expressing such compounds are contemplated aspossible vaccine components. Interleukins and cytokines, include but arenot limited to interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18,-interferon, -interferon, -interferon, angiostatin, thrombospondin,endostatin, GM-CSF, G-CSF, M-CSF, METH 1, METH 2, tumor necrosis factor,TGF, LT and combinations thereof.

3. Chemokines

Chemokines, nucleic acids that encode for chemokines, and/or cells thatexpress such also may be used as vaccine components. Chemokinesgenerally act as chemoattractants to recruit immune effector cells tothe site of chemokine expression. It may be advantageous to express aparticular chemokine coding sequence in combination with, for example, acytokine coding sequence, to enhance the recruitment of other immunesystem components to the site of treatment. Such chemokines include, forexample, RANTES, MCAF, MIP1-alpha, MIP1-Beta, IP-10 and combinationsthereof. The skilled artisan will recognize that certain cytokines arealso known to have chemoattractant effects and could also be classifiedunder the term chemokines.

4. Immunogenic Carrier Proteins

In certain embodiments, an antigenic composition may be chemicallycoupled to a carrier or recombinantly expressed with a immunogeniccarrier peptide or polypetide (e.g., a antigen-carrier fusion peptide orpolypeptide) to enhance an immune reaction. Exemplary and preferredimmunogenic carrier amino acid sequences include hepatitis B surfaceantigen, keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).Other albumins such as ovalbumin, mouse serum albumin or rabbit serumalbumin also can be used as immunogenic carrier proteins. Means forconjugating a polypeptide or peptide to a immunogenic carrier proteinare well known in the art and include, for example, glutaraldehyde, mmaleimidobenzoyl N hydroxysuccinimide ester, carbodiimide and bisbiazotized benzidine.

5. Biological Response Modifiers

It may be desirable to coadminister biologic response modifiers (BRM),which have been shown to upregulate T cell immunity or downregulatesuppressor cell activity. Such BRMs include, but are not limited to,cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.); low dosecyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, N.J.), or a geneencoding a protein involved in one or more immune helper functions, suchas B 7.

6. Adjuvants

Immunization protocols have used adjuvants to stimulate responses formany years, and as such adjuvants are well known to one of ordinaryskill in the art. Some adjuvants affect the way in which antigens arepresented. For example, the immune response is increased when proteinantigens are precipitated by alum. Emulsification of antigens alsoprolongs the duration of antigen presentation.

In one aspect, an adjuvant effect is achieved by use of an agent, suchas alum, used in about 0.05 to about 0.1% solution in phosphate bufferedsaline. Alternatively, the antigen is made as an admixture withsynthetic polymers of sugars (Carbopol®) used as an about 0.25%solution. Adjuvant effect may also be made my aggregation of the antigenin the vaccine by heat treatment with temperatures ranging between about70° to about 101° C. for a 30 second to 2 minute period, respectively.Aggregation by reactivating with pepsin treated (Fab) antibodies toalbumin, mixture with bacterial cell(s) such as C. parvum, an endotoxinor a lipopolysaccharide component of Gram negative bacteria, emulsion inphysiologically acceptable oil vehicles, such as mannide mono oleate(Aracel A), or emulsion with a 20% solution of a perfluorocarbon(Fluosol DA®) used as a block substitute, also may be employed.

Some adjuvants, for example, certain organic molecules obtained frombacteria, act on the host rather than on the antigen. An example ismuramyl dipeptide (N acetylmuramyl L alanyl D isoglutamine [MDP]), abacterial peptidoglycan. The effects of MDP, as with most adjuvants, arenot fully understood. MDP stimulates macrophages but also appears tostimulate B cells directly. The effects of adjuvants, therefore, are notantigen specific. If they are administered together with a purifiedantigen, however, they can be used to selectively promote the responseto the antigen.

Adjuvants have been used experimentally to promote a generalizedincrease in immunity against unknown antigens (e.g., U.S. Pat. No.4,877,611).

In certain embodiments, hemocyanins and hemoerythrins may also be usedin the invention. The use of hemocyanin from keyhole limpet (KLH) ispreferred in certain embodiments, although other molluscan and arthropodhemocyanins and hemoerythrins may be employed.

Various polysaccharide adjuvants may also be used. For example, the useof various pneumococcal polysaccharide adjuvants on the antibodyresponses of mice has been described (Yin et al., 1989). The doses thatproduce optimal responses, or that otherwise do not produce suppression,should be employed as indicated (Yin et al., 1989). Polyamine varietiesof polysaccharides are particularly preferred, such as chitin andchitosan, including deacetylated chitin.

Another group of adjuvants are the muramyl dipeptide (MDP, Nacetylmuramyl L alanyl D isoglutamine) group of bacterialpeptidoglycans. Derivatives of muramyl dipeptide, such as the amino acidderivative threonyl-MDP, and the fatty acid derivative MTPPE, are alsocontemplated.

U.S. Pat. No. 4,950,645 describes a lipophilic disaccharide-tripeptidederivative of muramyl dipeptide which is described for use in artificialliposomes formed from phosphatidyl choline and phosphatidyl glycerol. Itis the to be effective in activating human monocytes and destroyingtumor cells, but is non-toxic in generally high doses. The compounds ofU.S. Pat. No. 4,950,645 and PCT Patent Application WO 91/16347, arecontemplated for use with cellular carriers and other embodiments of thepresent invention.

Another adjuvant contemplated for use in the present invention is BCG.BCG (bacillus Calmette-Guerin, an attenuated strain of Mycobacterium)and BCG cell wall skeleton (CWS) may also be used as adjuvants in theinvention, with or without trehalose dimycolate. Trehalose dimycolatemay be used itself. Trehalose dimycolate administration has been shownto correlate with augmented resistance to influenza virus infection inmice (Azuma et al., 1988). Trehalose dimycolate may be prepared asdescribed in U.S. Pat. No. 4,579,945.

BCG is an important clinical tool because of its immunostimulatoryproperties. BCG acts to stimulate the reticulo-endothelial system,activates natural killer cells and increases proliferation ofhematopoietic stem cells. Cell wall extracts of BCG have proven to haveexcellent immune adjuvant activity. Molecular genetic tools and methodsfor mycobacteria have provided the means to introduce foreign genes intoBCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990;Martin et al., 1990).

Live BCG is an effective and safe vaccine used worldwide to preventtuberculosis. BCG and other mycobacteria are highly effective adjuvants,and the immune response to mycobacteria has been studied extensively.With nearly 2 billion immunizations, BCG has a long record of safe usein man (Luelmo, 1982; Lotte et al., 1984). It is one of the few vaccinesthat can be given at birth, it engenders long-lived immune responseswith only a single dose, and there is a worldwide distribution networkwith experience in BCG vaccination. An exemplary BCG vaccine is sold asTICE BCG (Organon Inc., West Orange, N.J.).

In a typical practice of the present invention, cells of Mycobacteriumbovis-BCG are grown and harvested by methods known in the art. Forexample, they may be grown as a surface pellicle on a Sauton medium orin a fermentation vessel containing the dispersed culture in a Dubosmedium (Dubos et al., 1947; Rosenthal, 1937). All the cultures areharvested after 14 days incubation at about 37° C. Cells grown as apellicle are harvested by using a platinum loop whereas those from thefermenter are harvested by centrifugation or tangential-flow filtration.The harvested cells are resuspended in an aqueous sterile buffer medium.A typical suspension contains from about 2×10¹⁰ cells/ml to about 2×10¹²cells/ml. To this bacterial suspension, a sterile solution containing aselected enzyme which will degrade the BCG cell covering material isadded. The resultant suspension is agitated such as by stirring toensure maximal dispersal of the BCG organisms. Thereafter, a moreconcentrated cell suspension is prepared and the enzyme in theconcentrate removed, typically by washing with an aqueous buffer,employing known techniques such as tangential-flow filtration. Theenzyme-free cells are adjusted to an optimal immunological concentrationwith a cryoprotectant solution, after which they are filled into vials,ampoules, etc., and lyophilized, yielding BCG vaccine, which uponreconstitution with water is ready for immunization.

Amphipathic and surface active agents, e.g., saponin and derivativessuch as QS21 (Cambridge Biotech), form yet another group of adjuvantsfor use with the immunogens of the present invention. Nonionic blockcopolymer surfactants (Rabinovich et al., 1994; Hunter et al., 1991) mayalso be employed. Oligonucleotides are another useful group of adjuvants(Yamamoto et al., 1988). Quil A and lentinen are other adjuvants thatmay be used in certain embodiments of the present invention.

One group of adjuvants preferred for use in the invention are thedetoxified endotoxins, such as the refined detoxified endotoxin of U.S.Pat. No. 4,866,034. These refined detoxified endotoxins are effective inproducing adjuvant responses in mammals. Of course, the detoxifiedendotoxins may be combined with other adjuvants to preparemulti-adjuvant-incorporated cells. For example, combination ofdetoxified endotoxins with trehalose dimycolate is particularlycontemplated, as described in U.S. Pat. No. 4,435,386. Combinations ofdetoxified endotoxins with trehalose dimycolate and endotoxicglycolipids is also contemplated (U.S. Pat. No. 4,505,899), as iscombination of detoxified endotoxins with cell wall skeleton (CWS) orCWS and trehalose dimycolate, as described in U.S. Pat. Nos. 4,436,727,4,436,728 and 4,505,900. Combinations of just CWS and trehalosedimycolate, without detoxified endotoxins, is also envisioned to beuseful, as described in U.S. Pat. No. 4,520,019.

In other embodiments, the present invention contemplates that a varietyof adjuvants may be employed in the membranes of cells, resulting in animproved immunogenic composition. The only requirement is, generally,that the adjuvant be capable of incorporation into, physical associationwith, or conjugation to, the cell membrane of the cell in question.Those of skill in the art will know the different kinds of adjuvantsthat can be conjugated to cellular vaccines in accordance with thisinvention and these include alkyl lysophosphilipids (ALP); BCG; andbiotin (including biotinylated derivatives) among others. Certainadjuvants particularly contemplated for use are the teichoic acids fromGram cells. These include the lipoteichoic acids (LTA), ribitol teichoicacids (RTA) and glycerol teichoic acid (GTA). Active forms of theirsynthetic counterparts may also be employed in connection with theinvention (Takada et al., 1995a).

Various adjuvants, even those that are not commonly used in humans, maystill be employed in animals, where, for example, one desires to raiseantibodies or to subsequently obtain activated T cells. The toxicity orother adverse effects that may result from either the adjuvant or thecells, e.g., as may occur using non irradiated tumor cells, isirrelevant in such circumstances.

One group of adjuvants preferred for use in some embodiments of thepresent invention are those that can be encoded by a nucleic acid (e.g.,DNA or RNA). It is contemplated that such adjuvants may be encoded in anucleic acid (e.g., an expression vector) encoding the antigen, or in aseparate vector or other construct. These nucleic acids encoding theadjuvants can be delivered directly, such as for example with lipids orliposomes.

7. Excipients, Salts and Auxiliary Substances

An antigenic composition of the present invention may be mixed with oneor more additional components (e.g., excipients, salts, etc.) which arepharmaceutically acceptable and compatible with at least one activeingredient (e.g., antigen). Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol and combinations thereof.

An antigenic composition of the present invention may be formulated intothe vaccine as a neutral or salt form. A pharmaceutically acceptablesalt, includes the acid addition salts (formed with the free aminogroups of the peptide) and those which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acid, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. A salt formedwith a free carboxyl group also may be derived from an inorganic basesuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxide, and such organic bases as isopropylamine, trimethylamine, 2ethylamino ethanol, histidine, procaine, and combinations thereof.

In addition, if desired, an antigentic composition may comprise minoramounts of one or more auxiliary substances such as for example wettingor emulsifying agents, pH buffering agents, etc. which enhance theeffectiveness of the antigenic composition or vaccine.

P. Vaccine Preparations

Once produced, synthesized and/or purified, an antigen or other vaccinecomponent may be prepared as a vaccine for administration to a patient.The preparation of a vaccine is generally well understood in the art, asexemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231,4,599,230, and 4,596,792, all incorporated herein by reference. Suchmethods may be used to prepare a vaccine comprising an antigeniccomposition comprising one or more antigens of Ureaplasma as activeingredient(s), in light of the present disclosure. In preferredembodiments, the compositions of the present invention are prepared tobe pharmacologically acceptable vaccines.

Pharmaceutical vaccine compositions of the present invention comprise aneffective amount of one or more Ureaplasma antigens or additional agentdissolved or dispersed in a pharmaceutically acceptable carrier. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of anpharmaceutical composition that contains at least one Ureaplasma antigenor additional active ingredient will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). The Ureaplasma vaccine may comprise different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. Except insofar as any conventional carrieris incompatible with the active ingredient, its use in the therapeuticor pharmaceutical compositions is contemplated.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The Ureaplasma vaccine may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the Ureaplasma vaccine is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, and may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

V. Exemplary Vaccine Administration

The manner of administration of a vaccine may be varied widely. Any ofthe conventional methods for administration of a vaccine are applicable.For example, a vaccine may be conventionally administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, rectally,nasally, topically, in eye drops, locally, using aerosol, injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

A vaccination schedule and dosages may be varied on a patient by patientbasis, taking into account, for example, factors such as the weight andage of the patient, the type of disease being treated, the severity ofthe disease condition, previous or concurrent therapeutic interventions,the manner of administration and the like, which can be readilydetermined by one of ordinary skill in the art.

A vaccine is administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. For example, the intramuscular route may be preferred inthe case of toxins with short half lives in vivo. The quantity to beadministered depends on the subject to be treated, including, e.g., thecapacity of the individual's immune system to synthesize antibodies, andthe degree of protection desired. The dosage of the vaccine will dependon the route of administration and will vary according to the size ofthe host. Precise amounts of an active ingredient required to beadministered depend on the judgment of the practitioner. In certainembodiments, pharmaceutical compositions may comprise, for example, atleast about 0.1% of an active compound. In other embodiments, the anactive compound may comprise between about 2% to about 75% of the weightof the unit, or between about 25% to about 60%, for example, and anyrange derivable therein However, a suitable dosage range may be, forexample, of the order of several hundred micrograms active ingredientper vaccination. In other non-limiting examples, a dose may alsocomprise from about 1 microgram/kg/body weight, about 5microgram/kg/body weight, about 10 microgram/kg/body weight, about 50microgram/kg/body weight, about 100 microgram/kg/body weight, about 200microgram/kg/body weight, about 350 microgram/kg/body weight, about 500microgram/kg/body weight, about 1 milligram/kg/body weight, about 5milligram/kg/body weight, about 10 milligram/kg/body weight, about 50milligram/kg/body weight, about 100 milligram/kg/body weight, about 200milligram/kg/body weight, about 350 milligram/kg/body weight, about 500milligram/kg/body weight, to about 1000 mg/kg/body weight or more pervaccination, and any range derivable therein. In non-limiting examplesof a derivable range from the numbers listed herein, a range of about 5mg/kg/body weight to about 100 mg/kg/body weight, about 5microgram/kg/body weight to about 500 milligram/kg/body weight, etc.,can be administered, based on the numbers described above. A suitableregime for initial administration and booster administrations (e.g.,innoculations) are also variable, but are typified by an initialadministration followed by subsequent inoculation(s) or otheradministration(s).

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1 5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies.

The course of the immunization may be followed by assays for antibodiesfor the supernatant antigens. The assays may be performed by labelingwith conventional labels, such as radionuclides, enzymes, fluorescents,and the like. These techniques are well known and may be found in a widevariety of patents, such as U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064, as illustrative of these types of assays. Other immune assayscan be performed and assays of protection from challenge with theUreaplasma vaccine can be performed, following immunization.

A. Enhancement of an Immune Response

The present invention includes a method of enhancing the immune responsein a subject comprising the steps of contacting one or more lymphocyteswith an Ureaplasma antigenic composition. In certain embodiments the oneor more lymphocytes is comprised in an animal, such as a human. In otherembodiments, the lymphocyte(s) may be isolated from an animal or from atissue (e.g., blood) of the animal. In certain preferred embodiments,the lymphocyte(s) are peripheral blood lymphocyte(s). In certainembodiments, the one or more lymphocytes comprise a T-lymphocyte or aB-lymphocyte. In a particularly preferred facet, the T-lymphocyte is acytotoxic T-lymphocyte.

The enhanced immune response may be an active or a passive immuneresponse. Alternatively, the response may be part of an adoptiveimmunotherapy approach in which lymphocyte(s) are obtained with from ananimal (e.g., a patient), then pulsed with composition comprising anantigenic composition. In a preferred embodiment, the lymphocyte(s) maybe administered to the same or different animal (e.g., same or differentdonors).

B. Cytotoxic T Lymphocytes

In certain embodiments, T-lymphocytes are specifically activated bycontact with an antigenic composition of the present invention. Incertain embodiments, T-lymphocytes are activated by contact with anantigen presenting cell that is or has been in contact with an antigeniccomposition of the invention.

T cells express a unique antigen binding receptor on their membrane (Tcell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines, that are crucial for the activation of B cells, T cytotoxiccells, macrophages and other cells of the immune system. In contrast, aT cytotoxic cells that recognizes an antigen MHC complex proliferatesand differentiates into an effector cell called a cytotoxic T lymphocyte(CTL). CTLs eliminate cells of the body displaying antigen by producingsubstances that result in cell lysis.

CTL activity can be assessed by methods described herein or as would beknown to one of skill in the art. For example, CTLs may be assessed infreshly isolated peripheral blood mononuclear cells (PBMC), in aphytohaemaglutinin stimulated IL 2 expanded cell line established fromPBMC (Bernard et al., 1998) or by T cells isolated from a previouslyimmunized subject and restimulated for 6 days with DC infected with anadenovirus vector containing antigen using standard 4 h ⁵¹Cr releasemicrotoxicity assays. In another fluorometric assay developed fordetecting cell mediated cytotoxicity, the fluorophore used is the nontoxic molecule alamarBlue (Nociari et al., 1998). The alamarBlue isfluorescently quenched (i.e., low quantum yield) until mitochondrialreduction occurs, which then results in a dramatic increase in thealamarBlue fluorescence intensity (i.e., increase in the quantum yield).This assay is reported to be extremely sensitive, specific and requiresa significantly lower number of effector cells than the standard ⁵¹Crrelease assay.

In certain aspects, T helper cell responses can be measured by in vitroor in vivo assay with peptides, polypeptides or proteins. In vitroassays include measurement of a specific cytokine release by enzyme,radioisotope, chromaphore or fluorescent assays. In vivo assays includedelayed type hypersensitivity responses called skin tests, as would beknown to one of ordinary skill in the art.

C. Antigen Presenting Cells

In general, the term “antigen presenting cell” can be any cell thataccomplishes the goal of the invention by aiding the enhancement of animmune response (i.e., from the T-cell or -B-cell arms of the immunesystem) against an antigen (e.g., a Ureaplasma antigen or aimmunologically functional equivalent) or antigenic composition of thepresent invention. Such cells can be defined by those of skill in theart, using methods disclosed herein and in the art. As is understood byone of ordinary skill in the art (see for example Kuby, 1993,incorporated herein by reference), and used herein certain embodiments,a cell that displays or presents an antigen normally or preferentiallywith a class II major histocompatability molecule or complex to animmune cell is an “antigen presenting cell.” In certain aspects, a cell(e.g., an APC cell) may be fused with another cell, such as arecombinant cell or a tumor cell that expresses the desired antigen.Methods for preparing a fusion of two or more cells is well known in theart, such as for example, the methods disclosed in Goding, pp. 65 66,71-74 1986; Campbell, pp. 75 83, 1984; Kohler and Milstein, 1975; Kohlerand Milstein, 1976, Gefter et al., 1977, each incorporated herein byreference. In some cases, the immune cell to which an antigen presentingcell displays or presents an antigen to is a CD4+TH cell. Additionalmolecules expressed on the APC or other immune cells may aid or improvethe enhancement of an immune response. Secreted or soluble molecules,such as for example, immunomodulators and adjuvants, may also aid orenhance the immune response against an antigen. Such molecules are wellknown to one of skill in the art, and various examples are describedherein.

D. Antibody Generation

In certain embodiments, isolated antibodies to the antigeniccompositions of the present invention are contemplated as useful forpurification, diagnostic and therapeutic applications. For example, itis contemplated that an antibody may be used as a vaccine component tobind a Ureaplasma antigen. As used herein, the term “antibody” isintended to refer broadly to any immunologic binding agent such as IgG,IgM, IgA, IgD and IgE. Generally, IgG or IgM are preferred because theyare the most common antibodies in the physiological situation andbecause they are most easily made in a laboratory setting. The term“antibody” is used to refer to any antibody like molecule that has anantigen binding region, and includes antibody fragments such as Fab′,Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (single chainFv), and the like. The techniques for preparing and using variousantibody based constructs and fragments are well known in the art. Meansfor preparing and characterizing an antibody are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; and Antibody Engineering, Second Edition, OxfordUniversity Press, 1995, each incorporated herein by reference).

In certain embodiment, one or more “humanized” antibodies are alsocontemplated, as are antibodies comprising components from variousorigins, such as for example, one or more chimeric antibodies frommouse, rat, or other species, bearing one or more human constant and/orvariable region domains; one or more bispecific antibodies; or one ormore recombinant and engineered antibodies and/or fragment(s) thereof.Methods for the development of one or more antibodies that are “customtailored” to a patient's disease are likewise known and such customtailored antibodies are also contemplated.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large scale production, and their use isgenerally preferred. MAbs may be readily prepared through use of wellknown techniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference.

In certain diagnostic or vaccine component purification aspects, anantibody one or more vaccine components, such as a Ureaplasma antigen,may be used. Non-limiting examples of such immunodetection methodsinclude enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescentassay, bioluminescent assay, and Western blot to mention a few. Thesteps of various useful immunodetection methods have been described inthe scientific literature, such as, e.g., Doolittle M H and Ben-Zeev O,1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamuraet al., 1987, each incorporated herein by reference. Often, the antibodymay be conjugated with an imaging agent to enhance detection of avaccine component ligand bound to the antibody, as would be known to oneof ordinary skill in the art. Many appropriate imaging agents are knownin the art, as are methods for their attachment to antibodies (see, fore.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509, eachincorporated herein by reference).

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. Patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

VI. Proteins, Polypeptides, and Peptides

The present invention also provides purified, and in preferredembodiments, substantially purified, Ureaplasma proteins, polypeptides,or peptides. The term “purified proteins, polypeptides, or peptides” asused herein, is intended to refer to an proteinaceous composition,isolatable from mammalian cells or recombinant host cells, wherein theat least one protein, polypeptide, or peptide is purified to any degreerelative to its naturally obtainable state, i.e., relative to its puritywithin a cellular extract. A purified protein, polypeptide, or peptidetherefore also refers to a wild type or mutant protein, polypeptide, orpeptide free from the environment in which it naturally occurs.

The nucleotide and protein, polypeptide and peptide sequences forvarious genes have been previously disclosed, and may be found atcomputerized databases known to those of ordinary skill in the art. Onesuch database is the National Center for Biotechnology Information'sGenBank® and GenPept databases. The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor by any technique that would be know to those of ordinary skill in theart. Additionally, peptide sequences may be synthesized by methods knownto those of ordinary skill in the art, such as peptide synthesis usingautomated peptide synthesis machines, such as those available fromApplied Biosystems (Foster City, Calif.).

Generally, “purified” will refer to a specific protein, polypeptide, orpeptide composition that has been subjected to fractionation to removevarious other proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bythe protein assays, as described herein below, or as would be known toone of ordinary skill in the art for the desired protein, polypeptide orpeptide.

Where the term “substantially purified” is used, this will refer to acomposition in which the specific protein, polypeptide, or peptide formsthe major component of the composition, such as constituting about 50%of the proteins in the composition or more. In preferred embodiments, asubstantially purified protein will constitute more than 60%, 70%, 80%,90%, 95%, 99% or even more of the proteins in the composition.

A peptide, polypeptide or protein that is “purified to homogeneity,” asapplied to the present invention, means that the peptide, polypeptide orprotein has a level of purity where the peptide, polypeptide or proteinis substantially free from other proteins and biological components. Forexample, a purified peptide, polypeptide or protein will often besufficiently free of other protein components so that degradativesequencing may be performed successfully.

Various methods for quantifying the degree of purification of proteins,polypeptides, or peptides will be known to those of skill in the art inlight of the present disclosure. These include, for example, determiningthe specific protein activity of a fraction, or assessing the number ofpolypeptides within a fraction by gel electrophoresis.

To purify a desired protein, polypeptide, or peptide a natural orrecombinant composition comprising at least some specific proteins,polypeptides, or peptides will be subjected to fractionation to removevarious other components from the composition. In addition to thosetechniques described in detail herein below, various other techniquessuitable for use in protein purification will be well known to those ofskill in the art. These include, for example, precipitation withammonium sulfate, PEG, antibodies and the like or by heat denaturation,followed by centrifugation; chromatography steps such as ion exchange,gel filtration, reverse phase, hydroxylapatite, lectin affinity andother affinity chromatography steps; isoelectric focusing; gelelectrophoresis; and combinations of such and other techniques.

Another example is the purification of a specific fusion protein using aspecific binding partner. Such purification methods are routine in theart. As the present invention provides DNA sequences for the specificproteins, any fusion protein purification method can now be practiced.This is exemplified by the generation of an specific protein glutathioneS transferase fusion protein, expression in E. coli, and isolation tohomogeneity using affinity chromatography on glutathione agarose or thegeneration of a polyhistidine tag on the N or C terminus of the protein,and subsequent purification using Ni affinity chromatography. However,given many DNA and proteins are known, or may be identified andamplified using the methods described herein, any purification methodcan now be employed.

Although preferred for use in certain embodiments, there is no generalrequirement that the protein, polypeptide, or peptide always be providedin their most purified state. Indeed, it is contemplated that lesssubstantially purified protein, polypeptide or peptide, which arenonetheless enriched in the desired protein compositions, relative tothe natural state, will have utility in certain embodiments.

Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein. Inactive products also have utility incertain embodiments, such as, e.g., in determining antigenicity viaantibody generation.

VII. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more vaccines of the invention or additionalagent dissolved or dispersed in a pharmaceutically acceptable carrier.The phrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one Ureaplasma vaccineor additional active ingredient will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The composition may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle composition that include the composition,one or more lipids, and an aqueous solvent. As used herein, the term“lipid” will be defined to include any of a broad range of substancesthat is characteristically insoluble in water and extractable with anorganic solvent. This broad class of compounds are well known to thoseof skill in the art, and as the term “lipid” is used herein, it is notlimited to any particular structure. Examples include compounds whichcontain long-chain aliphatic hydrocarbons and their derivatives. A lipidmay be naturally occurring or synthetic (i.e., designed or produced byman). However, a lipid is usually a biological substance. Biologicallipids are well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glycolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof. Of course, compounds other than those specifically describedherein that are understood by one of skill in the art as lipids are alsoencompassed by the compositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the composition may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the composition isformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, the composition may be administered via aparenteral route. As used herein, the term “parenteral” includes routesthat bypass the alimentary tract. Specifically, the pharmaceuticalcompositions disclosed herein may be administered for example, but notlimited to intravenously, intradermally, intramuscularly,intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S.Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and5,399,363 (each specifically incorporated herein by reference in itsentirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compound maybe formulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

VIII. Detection of Ureaplasma

In some embodiments of the invention, compositions of the invention areutilized for detection of Ureaplasma. In specific cases, for example,anti-MBA monoclonal antibody is employed for detection of Ureaplasma. Inspecific embodiments, antibody against the MBA antigen (for example, theconserved portion or 5′ end of the MBA antigen) is utilized to identifythe organism as being present in culture, serum and/or other bodyfluids. In certain aspects, antibody against part of SEQ ID NO:4 isemployed, for example.

Upon detection of Ureaplasma in a culture or in an individual, therespective culture or individual may be treated with one or moretherapeutic compositions of the invention and/or other therapeuticmeans, including antibiotics, for example.

The skilled artisan recognizes that there are routine methods in the artfor obtaining a sample to assay for detection of Ureaplasma.

IX. Exemplary Kits of the Invention

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, a Ureaplasma immunogenic composition may becomprised in a kit, including a vaccine, for example a DNA vaccine.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit, the kitalso will generally contain a second, third or other additionalcontainer into which the additional components may be separately placed.However, various combinations of components may be comprised in a vial.The kits of the present invention also will typically include a meansfor containing the composition and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow molded plastic containers into which the desired vials areretained.

Therapeutic kits of the present invention include kits comprising achemical compound or pharmaceutically acceptable salts thereof or aprotein, polypeptide, peptide, inhibitor, gene, vector and/or otherimmunological effector. Such kits may generally contain, in suitablecontainer means, a pharmaceutically acceptable formulation of a multiplebanded antigen chemical compound or pharmaceutically acceptable saltsthereof or protein, polypeptide, peptide, domain, inhibitor, and/or agene and/or vector expressing any of the foregoing in a pharmaceuticallyacceptable formulation. The kit may have a single container means,and/or it may have distinct container means for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The compositions may alsobe formulated into a syringeable composition. In which case, thecontainer means may itself be a syringe, pipette, and/or other such likeapparatus, from which the formulation may be applied to an infected areaof the body, injected into an animal, and/or even applied to and/ormixed with the other components of the kit. However, the components ofthe kit may be provided as dried powder(s). When reagents and/orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Perinatal Ureaplasma Infection and Brain Development

Utilizing a published murine model for chorioamnionitis, e13.5 dayfetuses are infected with 5000 ccu of Ureaplasma or saline by directinfection. At e17.5 days, Ureaplasma's impact is evaluated between thegroups on placental histopathology, blood, amniotic fluid, placentaUreaplasma culture and PCR, and inflammatory mediators. There is alsoevaluation for evidence for Ureaplasma brain infection an inflammationon day e17.5, 6, and 18 wks after delivering including brain: Ureaplasmaculture and PCR; inflammatory mediators; histopathology andhistochemistry. Placenta histopathology is read by an expert in placentapathology in a blinded fashion. One then evaluates pups born to motherswith Ureaplasma induced chorioamnionitis vs. pups exposed to salinein-utero for development of a neurologic or developmental phenotypeduring the 18 week observation. One employs standardized neurologic anddevelopmental examinations by individuals blinded to the group. In thissame chorioamnionitis model, one can determine the impact of a prenatalrDNA Ureaplasma vaccine on development of brain inflammation, andneurologic and developmental phenotypes by individuals blinded to thetreatment group assignment (rDNA Ureaplasma or saline vaccine).

Example 2 Development of a Ureaplasma DNA Vaccine for the Treatment ofMammalian Infection

Methods and Exemplary Data: The inventors have developed the methods tocreate an Ureaplasma vaccine and evaluate the efficacy and itssubsequent antibody production. Specifically: Vaccine Development: Indeveloping this DNA vaccine the Ureaplasma gene of interest was clonedand inserted into a pVAX1 vector. Briefly, the DNA fragment responsiblefor the multiple antigen binding (MAB) region of Ureaplasma serotype 6(386 bp) was generated by PCR with specific primers: sense (TG TTC ATATTT TTT ATC AG; SEQ ID NO:2); antisense (CCAAATGACCTTTTGTAACTAGTA; SEQID NO:3). In order to increase the efficacy of antigen expression, aKozak codon (ANNATGG; SEQ ID NO:1) was inserted at the beginning of thesense primer. The stop codon used was that provided in the vector (TAG).The DNA fragment from the PCR was then inserted into the vector pVAX1(Invitrogen). This vector contains an early CMV promotor and a bovinegrowth hormone polyadenylation. The plasmid vector containing theantigen gene was transformed into E. coli (DH5a), clones were selected,and grown in LB media. Plasmid DNA was purified with a Qiagen miniprepkit. The orientation of the insert was confirmed by enzyme digestion andthen the correct plasmid DNA was grown in LB media for injection. Eachkit allowed us to isolate 1 mg of pDNA.

ELISA Assay (Whole Bacteria): This was performed as previously reported(Echahidi et al., 2001) with modifications. In brief: Ureaplasmareference strains were grown in 10 ml of 10 B broth to 10⁶ ccu/ml.Organisms were centrifuged at 25,000×g for 30 min at 4° C. andharvested. The pellet was washed thrice with phosphate-buffered saline(PBS), the final pellet resuspended in 100 μl of PBS, and diluted withmethanol to 10 ml. To coat the microtiter plates, 100 μl of the antigenpreparation diluted in methanol was added to each well and incubated atroom temperature overnight until complete methanol evaporation. Thewells were saturated with bovine serum albumin (3% [wt/vol]) in PBS andwashed twice.

Washing was performed with PBS containing 0.1% Tween 20. 100 ul of mouseserum diluted 1:2 in PBS was added to the wells and incubated for 1 hrat room temperature. After a wash, 100 μl of horseradishperoxidase-labeled polyclonal anti-mouse immunoglobulin diluted in PBScontaining 0.05% Tween 20 was added to the wells and the mixtures wereincubated for 30 min at room temperature. After a wash, the peroxidasesubstrate (o-phenylenediamine) was added to the wells and the mixtureswere incubated for 15 min in the dark at room temperature. The substratereaction was stopped by adding H₂SO₄ (4 N), and optical density (OD)measured at 490 nm. Negative controls were obtained by testing theconjugate without adding serum. The blank wells received ELISA reagentsbut no antigens or serum. Standard statistical methods were used toevaluate the data. Results: The serum level of antibody againstUreaplasma was detected using an ELISA with a clinical strain forserotype 14. Optical density increased from 1.0 to greater than 3.3.

Immunization of Mice: Adult female FVB white mice were injectedintraperitoneal (IP) with 500 ug per dose of the vaccine with differentschedules. Group 1 received vaccine day 0 and 11 wks. Group 2 receivedvaccine day 0, 4 wks, and 11 wks. Blood was collected from thevaccinated mice every 2 wks after the first vaccination and serumisolated.

Bacterial Killing Assay: To determine if the antibody generated by thevaccine participated in bacterial killing, we performed a previouslyreported neutrophil-mediated bacterial killing assay (Weisman andLorenzetti, 1989) with modifications: In summary, we used 20 ul of 10⁷cells/ml of neutrophils which were isolated from healthy donors (GulfCoast Blood Center, Houston Tex.), 20 ul of 10⁶ ccu/ml serotype 14Ureaplasma, 10 ul of human complement sera (Sigma, S1764 pre-adsorbedwith S. epidermidis) diluted 1:4, 20 l of heat inactivated mouse serum(as an antibody source) collected at 12 wks after first vaccineinjection, and qs the final well volume to 200 μl with 10B broth.Bacteria, serum, complement, and neutrophils were added together inmicrotiter plate wells, sealed and incubated at 37° C. for 5 days. Otherwells contained bacteria alone, bacteria and serum, bacteria andneutrophils, bacteria and complement, bacteria with serum andneutrophils, bacteria with serum and complement, bacteria withcomplement and neutrophils. Control wells contained no bacteria witheach of the combinations above. The OD was read at 650 nm every 2 hoursin a microtiter plate reader for the first 48 hr and every 24 hr fornext 72 hours. The 10 B media is pH sensitive and converts from yellowto red with growth of the bacteria. Color change was also visuallyevaluated every 6 hrs. No change in color or absorbance was observedwith the negative controls. The Wilcoxon-rank sum test is used tocompare bacterial killing among the different groups at the differentdilutions. Results: Animals who received 500 ug/dose of the vaccine at0, 4 and 11 wks, were bled at 12 wks. Serum demonstrated evidence ofbacterial killing of a clinical strain of Ureaplasma serotype 14 ata >1:80 dilution.

Animal Models: Two animal models of Ureaplasma infection have beendeveloped in pups including a sepsis model (Kong et al., 2008) and abronchopulmonary dysplasia (BPD) model (Walls et al., 2009). Thevaccine's effect on the sepsis model has been evaluated. The BPD modelis more time consuming and expensive and will be evaluated when thevaccine is optimized.

In vivo Protection from Sepsis in Pups: All vaccinated mice were matedwith males at 12 wks after the first vaccination. Pups were delivered at15 wks. Pups were then infected with 2 doses of 0.1 cc of 10⁶ ccu/ml ofUreaplasma parvum clinical strain B079 serotype 14 at one day of age.Control litters were composed of pups born to unvaccinated dams andinfected with the same dose and strain of Ureaplasma. The survival ratewas calculated after 8 days and compared between litters born tovaccinated and unvaccinated dams. In Study #2, pregnancy was initiatedin the same dams at 17 wks. Pups were delivered at 21 wks and treatedsimilarly. Standard analyses of proportions were used to assess thestatistical significance. The results are described below:

Vaccine Doses: Two Three Control Study 1: N = 20 19 27 N Survival at 48hrs = 16 18 14 % Survival at 48 hrs = 80 95 52 P value vs control 0.0670.0027 Study 2: N = 8 13 52 N Survival at 48 hrs = 5 11 18 % Survival at48 hrs = 63 85 35 P value vs control 0.24 0.0016 Combining Study 1 and2: N = 28 32 79 N Survival at 48 hrs = 21 29 32 % Survival at 48 hrs =75 91 41 P value vs control 0.0021 0.000001

This DNA vaccine given to mice before pregnancy is effective inpreventing sepsis and death in pups of vaccinated dams for at least twoconsecutive pregnancies. It also is effective against an Ureaplasmastrain/serotype of infecting organism different then that from which thevaccine was developed, suggesting a broad range of efficacy.

Exemplary Studies:

Optimize vaccine design: A vaccine may be optimized to enhance itsefficacy and effectiveness for further development and use.Specifically, one could utilize one or more of the following changes indesign: 1) Add the MPV-CTE+rev to the current vaccine to increaseenvelope expression and immunogenicity. 2) Exchange the pVAX1 vectorwith the pVAX200-DEST vector in the vaccine plasmid. 3) Express morethan one immunogen in the plasmid to enhance the vaccines efficacy andimpact. Specifically express the multiple binding proteins (MBP) fromseveral if not all serotypes of Ureaplasma. 4) Add an N-terminalubiquitin signal to the stop code. 5) Add strings of MHC class Iepitopes from different pathogens or oligonucleotides (e.g. CpG motif)to the plasmid raise cytotoxic T-cell responses. 6) Add humangranulocyte-macrophage colony-stimulating factor (hGM-CSF) as an immuneenhancer to the plasmid to possibly further optimize the vaccine.

Optimize vaccine delivery: In light of the successful response to thevaccine we have observed to date, one can optimize the vaccine'sdelivery system to enhance its efficacy and effectiveness for furtherdevelopment and use. Thus one can characterize different deliverysystems. The most expensive and time consuming aspect of the vaccinesproduction is the amount of pDNA that must be produced, so one canreduce that while maintaining or increasing efficacy. Specifically, onecan decrease the dose of the vaccine to 0.2 to 200 ug. Utilizing thesmallest effective dose, one could investigate the delivery systemincluding: 1) Delivery of an aqueous solution using methods differentfrom the current IP system including IM, ID and SC routes. 2) Deliveryof gold or tungsten microparticles with vaccine absorbed onto theirsurface via the ED route. Since this organism is a sexually transmitteddisease, we would also investigate this method via the vaginal mucosa.The most effective delivery system will then need to be evaluated forimpact of the animal's age and sex on the immune response.

Evaluation of vaccine design and delivery optimization: To evaluate theproposed vaccine design and delivery changes we will use a series ofmethods including: 1) Incorporation of tracer (e.g. LacZ) into thetarget cells in vitro and in vivo (see elsewhere herein for Methods). 2)Changes in the vaccinated animal's serum total antibody againstUreaplasma (of varying serotype) whole bacteria as detected by ELISA(see elsewhere herein). 3) Changes in vaccinated animal's serum specificantibody against Ureaplasma (of varying serotypes) MBP as detected byELISA (see below for Methods). 4) Changes in vaccinated animal's serumbacterial killing assay against live Ureaplasma (of varying serotypes)as detected in culture (see elsewhere herein). 6) Changes in thesurvival, bacteremia, and inflammation of infected pups of vaccinatedanimals in multiple models of Ureaplasma infection including sepsis (seeelsewhere herein), BPD (see elsewhere herein). 7) Changes in male orfemale mouse genitourinary and or gastrointestinal Ureaplasmacolonization. (see below Methods) 8) Changes in pregnancy outcomes (pupnumber, pup size, gestation length) of females if they or their malepartners or both are infected (see below Methods).

Incorporation of LacZ Tracer: This may be utilized as previouslydescribed in organisms (in-vitro) (Silva et al., 2009) and cells/tissue(in vivo) (Joussemet et al., 2005) and provide us with rapid feedback onthe changes made to both the vaccine's design and delivery.

ELISA Assay for MBA: This MBA assay is performed as previously reported(Vancutsem et al., 2008) with modifications. In brief: Recombinant MBA(rMBA) is produced from the serotype and strain of Ureaplasma used forthe vaccine to determine the amount of MBA specific antibody present inthe serum of vaccinated animals or the pups of vaccinated animals. TheMBA gene of interest is produced, cloned, expressed, purified andevaluated against animal serum. In addition one can characterize theantigen and the antibody (class and subclass) using standard immunologictechniques.

Bacterial Adherence Assay: This adherence assay is performed aspreviously reported (Smith et al., 1994; Thirkell et al., 1989;Torres-Morquecho et al., 2010) with modifications. In brief: A594 cellsobtained from the ATCC are grown in DMEM supplemented with 10% FBS andwithout antibiotics at 37° C. Ureaplasma in 10B broth is added at aconcentration of about 10⁵ ccu/ml to all wells of a six well plasticplate, serum at various dilutions are added to each well, and incubated.Control wells can contain 10 B media only and serum of non vaccinatedanimals or pups of these animals. All experiments are conducted induplicate.

Adherence of Ureaplasma is quantified using a colorimetric method(Bertholet assay) that monitors ammonia produced from urea by Ureaplasmaurease. The Mann-Whitney U test is used to evaluate the significance inadherence inhibition generated by the serum of vaccinated animals orpups of these animals.

Additional Animal Models: One can evaluate the impact of the vaccine onadditional adult male and female animal models that have yet to be madeoperational including at least: 1) Genitourinary and GastrointestinalColonization: This has previously been reported and one can adapt thesemethodologies. (Audring et al., 1989; Fun and Taylor-Robinson, 1993;Iwasaka et al., 1986) In short, estrogen (female) and testosterone(male) treated mice are given oral and or genitourinary inoculations ofUreaplasma and colonization is reported to be prolonged (>3 weeks) andvery heavy (>100 ccu/swab). 2) Infertility and Low Birth Weight: Thishas previously been reported and one can adapt these methodologies.(Audring et al., 1989; Engel et al., 1988; Swenson and O'Leary, 1978) Inshort: Colonized males and/or females are mated. The success of matingsin vaccinated versus non-vaccinated controls is calculated, birth weightmeasured, and the number of pups counted. Once these models areoperational, one can incorporate them into the evaluation of thevaccine's impact. One can initially use a pup sepsis and BPD model toassess the clinical outcome of vaccine intervention, but one cansubsequently employ the vaccine in these other models.

Organisms: Ureaplasma serotypes/strains are grown in 10 B broth beforeeach use from a frozen stock solution (5×10⁶ ccu/ml). The effectivenessof each vaccine is tested against each serovar of biovar parvum becauseit appears most significant and for key biovar urealyticum (Blanchard etal., 1990; Blanchard et al., 1993; Brown et al., 1981; Cassell et al.,1983) serovar.

Animals: FVB albino mice are used for all the animal experiments and fedantibiotic free water and food ad libitum. Pregnancies result from timeimpregnation at an animal facility. Pups are kept with dams throughouteach experiment. At 14 days surviving pups are either euthanized orweaned.

Example 3 Perinatal Ureaplasma Infection and Suckling Mouse BrainDevelopment

Ureaplasma have the following characteristics: small (0.1-0.85 um);devoid of a cell wall (insensitive to penicillin and gram stain); needurea for growth and produce urease (Pollack, 1986); not folic acidsynthesizers (not susceptible to sulfonamides or trimethoprim). The mostsensitive method of isolating Ureaplasma is inoculation in to liquidmedium, detection by urease activity, and subculturing to agar forcolony identification (Robertson, 1978; Taylor-Robinson, 1989;Taylor-Robinson et al., 1967; Taylor-Robison and Gourlay, 1984;Taylor-Robinson, 1989). Simple and rapid methods of Ureaplasmaidentification such as PCR have been developed and confirm culture(Abele-Horn et al., 1996; Blanchard et al., 1990; Blachard et al., 1993;Cunleffe et al., 1996; Willoughby et al., 1990), but commercial kits arenot yet readily available. The diagnosis of Ureaplasma infection iscomplicated by its lack of gram staining, fastidious nature, and needfor special growth and transport media. In view of these difficulties inidentification, unless Ureaplasma is anticipated it may escape detectionand that may explain the relative paucity of reports and clinicalexperience. PCR may remain positive for months and thus may onlyrepresent nonviable organisms (Cassell et al., 1983; Clifford et al.,2010). Thus, more laborious, complex identification methods arerequired, and only a few laboratories have that capability.

Ureaplasma attaches and invades a variety of cells (Busolo et al., 1984;Masover et al., 1977; Robertson et al., 1991; Saada et al., 1991;Shepard and Masover, 1979; Smith et al., 1994; Torres-Morquecho et al.,2010); is associated with cell apoptosis (Li et al., 2002); increasesinflammatory cytokines (McGarrity and Kotani, 1986; Smith et al., 1994;Torres-Morquecho et al., 2010).

Prevention or Treatment of Ureaplasma Infection: Eradication ofUreaplasma from the urogenital tracts of women and their partners hasbeen proposed. (Kundsin et al., 1996) However, Ureaplasma is notsusceptible in-vitro to penicillins, sulfonamides, trimethoprim,aminoglycosides, and clindamycin, but are generally (about 90%)susceptible in-vitro to tetracyclines, and variably to macrolides (e.g.erythromycin). (Cassell et al., 1993) In recent studies, these variablesusceptibilities. (Molina et al., 2010; Okunola et al., 2006; Okunola etal., 2007; Weisman et al., 2009) Prophylactic antibiotics at deliverydid not effect Ureaplasma colonization of the chorioamnion. (3)Macrolides (Eschenbvach, 1993; Mazor et al., 1993; Romero et al., 1993)have not been reliable eradicating genital tract Ureaplasma or adverseperinatal outcomes in two randomized controlled trials. In addition, incouples attending infertility clinics, genital tract Ureaplasmapersisted despite antibiotics. (Hipp et al., 1983) Although newerantibiotics (e.g. glycylcyclines (Kenny and Cartwright, 1994) andquinolones (Kenny and Cartwright, 1996)) may prove more effective,safety and efficacy during pregnancy are unproven. In view of the highcolonization and sexual transmission rates, and variable sensitivity ofUreaplasma, it is unlikely that current antibiotic strategies will beeffective in its eradication.

It has been suggested but not demonstrated that lack of specificantibody may be critical for preventing Ureaplasma infection, becausespecific antibody may inhibit growth in vitro. (Cassell et al., 1993)Hypogammaglobulinemic patients have an increased susceptibility toUreaplasma (Taylor-Robinson et al., 1986) and serological studies ofhypogammaglobulinemic patients (Volger et al., 1985), pre-term infants(Quinn et al., 1983), and women with recurrent spontaneous abortions(6.1) support this concept. The increased susceptibility of infants <30wks gestational age to Ureaplasma respiratory disease may be related tohypogammaglobulinemia (5) or lack of specific antibody (Cassell et al.,1988; Casell et al., 1988). It has been suggested but not demonstratedthat monoclonal antibodies to specific protein antigens of Ureaplasmacan inhibit growth of these organisms in-vitro and indicates thatspecific antibody may be important for host defense. (Watson et al.,1990)

Neurologic Impact of Ureaplasma: Considerable evidence links Ureaplasmarespiratory colonization with neonatal lung morbidity, but few studiesinvestigate intrauterine Ureaplasma with neurologic morbidities and theyare listed here. The risk of severe IVH (grade ≧3) was 2.5 fold higherin serum Ureaplasma PCR-positive (n=74) than PCR-negative infants(n=239) after adjustment for gestational age. (Viscardi et al., 2008) U.parvum was the species identified in all PCR-positive infants withsevere IVH. The risk for severe IVH increased to five fold in PCR

positive patients with elevated serum IL1β. Another (n=866) report(Olomu et al., 2009), observed that Ureaplasma in the placentaparenchyma before 28 weeks was associated with increased: preterm laborand delivery; fetal and maternal inflammation; intraventricularhemorrhage; echolucent brain lesions. These lesions predict motor andcognitive limitations and poor outcome. (Leviton et al., 1999) Anotherstudy observed Ureaplasma infection of the amniotic cavity at the timeof preterm birth (n=67) was associated at 2 years with: abnormal PDIscore (OR 3.1, CI 1.3-7.1); abnormal neurologic outcome (OR 4.8, CI1.7-13.8); higher probability of cerebral palsy (OR 4.8, Cl 1.4-16.4) vscontrol patients (n=47) with amniotic fluid negative for Ureaplasma,irrespective of gestational age or birthweight. (Berger 2009) Severalgroups have suggested that proinflammatory cytokines (e.g. IL-1β, IL-6,and TNF α) might be the link between perinatal infection and neonatalbrain damage. (Dammann et al., 1997; Kaukola et al., 2006) and one study(n=1078) of high risk patients, reported the timing and use ofantibiotics affected development of echolucent brain images (Leviton etal., 1999). Although more information is needed to assess Ureaplasma'scontribution to adverse neurodevelopment, Ureaplasma exposed infantsappear more severely affected neurologically.

The role for Ureaplasma in brain injury is supported in the only reportof a mouse model. In this model (Normann et al., 2009), intraamnioticinfection with Ureaplasma leads to inflammation and disturbed braindevelopment. Specifically, they observed: a decreased density ofcalbindin protein-positive and calretinin-positive neurons, suggesting adisturbed production, maturation, and or survival of interneurons, whichplay a key role in associative and cognitive functions (Mohler, 2007);decreased MBP staining which most likely reflects decreased myelinationwhich again has been associated with limited cognitive function (Back,2006), and increased central microglial activation which the authorsspeculated most likely participated in the effects on interneurons andmyelin. In certain aspects, these observations could have been due todirect spread and infection of the brain or secondary immune orinflammatory effects or both. A primate model (Novy et al., 2009)indicates both since they observed that intraamniotic infection withUreaplasma lead to a systemic inflammatory response and in someinstances cerebrospinal fluid cultures that contained Ureaplasma.Depending on the mechanism of the injury, treatments would vary.

There have been reports of Ureaplasma cultured from the brains ofinfants including two preterm infants who died of intraventricularhemorrhage (Ollikainen et al., 1993) and a neonate with a brain abscess.(Rao et al., 2002) There have been 72 cases reported of Ureaplasmameningitis. (Cassell et al., 1988) Seven prospective studies estimatedthe incidence of Ureaplasma in neonates presenting with clinicalsymptoms. In studies of 100 (Waites et al., 1988) and 313 (Viscardi,2010) and 66 (Sethi et al., 1999) preterm infants, CSF grew Ureaplasmain 8%, 19.1%, and 9% of patients respectively. In a study of 318neonates (only one preterm) born at a community hospital (Waites et al.,1990) and 69 neonates of variable gestation (Olomu et al., 2009), 1.6%and 1.5% grew Ureaplasma from their CSF respectively. The largest studyreported an incidence of 0.2% in 920 infants but the methods appearedflawed and insensitive (Waites et al., 1995). There are also seven casereports or small case series reporting Ureaplasma as a cause ofmeningitis. (Biran et al., 2010; Chung et al., 2007; Garland and Murton,1987; Hentschel et al., 1993; Neal et al., 1994; Singh et al., 2003;Stahelin-Massik et al., 1994) Of the reported patients with Ureaplasmameningitis, 86% were premature, 90% were in the first 2 weeks of life,90% were asymptomatic, and 72% were U. parvum. Although abnormalities inCSF cell count, glucose and protein are described, all or some of thesecan be absent in many cases. It also remains unclear whether Ureaplasmaenters the CSF via the blood (15% had positive blood cultures) ordirectly from the respiratory tract (33% had positive respiratorycultures) across the cribriform plate or both, or Ureaplasma's impact isaffected via inflammatory mediators or the infant's immune responsefollowing colonization in the perinatal period.

The inventors have developed the following: assays to identifyUreaplasma (physiologic, culture, and PCR) its biovars, serovars, andantibiotic sensitivity (Molina et al., 2010; Okunola et al., 2006;Okunola et al., 2007; Weisman et al., 2009); suckling mouse models toevaluate the affect of this organism and antibiotic treatment orprevention strategies in Sepsis (Kenny and Cartwright, 1996) and BPD(Walls et al., 2009). Most recently the inventors developed anUreaplasma rDNA vaccine.

Ureaplasma Vaccine Development: The portion of the MBA Ureaplasma genethat codes for a constant region across all serotypes was selected asthe target for vaccine and antibody development. Serotype 6 was selectedas the gene source because it is frequently an invasive clinicalserotype. (Vancustem et al., 2008) In developing this rDNA vaccine theUreaplasma gene of interest was cloned and inserted into a pVAX1 vector.A whole bacteria ELISA assay was performed as previously reported(Echahidi et al., 2001), with modifications, on serum samples from dams,and there were significant antibody levels (optical density increasedfrom 1.0→3.3) against a serotype 14 clinical strain of Ureaplasma withappropriate controls. A bacterial killing assay, as we previouslyreported (Weisman et al., 1989) with modifications, demonstratedevidence of bacterial killing at >1:80 dilution against a serotype 14clinical strain of Ureaplasma with appropriate controls. A sepsis model(Kong et al., 2008) as previously reported was used to evaluate in vivothe protection to the maternal vaccine provided pups. This rDNA vaccinegiven to mice before pregnancy was effective in preventing sepsis anddeath (91% vs 41%, p<0.000001) in pups of vaccinated dams for at leasttwo consecutive pregnancies, against an Ureaplasma infecting organism ofa different serotype (Casell et al., 1988) then that from which thevaccine was developed (Biran et al., 2010), indicating a broad efficacy.

Exemplary Study Design and Methodology:

To develop a mouse model of antenatal Ureaplasma chorloamnionitis, onecan utilize the method recently published (Normann et al., 2009) andsomewhat modified. In short: 1) one can mate female FVB white mice(Charles River, Wilmington, Mass.) with male C57 BL6 mice (CharlesRiver) to generate a pup Fl FVB:C57BL6 hybrid for study. One cangenerate the hybrid because FVB white mice can develop blindness by 6months of age and that would interfere with the developmental testing.The FVB mouse is utilized, in certain cases, because our Ureaplasmaanimal investigations have been conducted in these mice. 2) a clinicalstrain of Ureaplasma serotype 14 are grown in selective media from afrozen aliquot, 2) embryonic day (e) 13.5, pregnant FVB white mice rerandomly allocated to one of two intraamniotic fluid injectionsubstances: a) saline injection, b) Ureaplasma injection (5000 ccu). Onecan inject Ureaplasma in saline, without media, to eliminate thepotential inflammatory effects of the media previously reported (Normannet al., 2009). Under sterile conditions, pregnant dams are anesthetizedwith isoflurane. The uterus externalized through a 12 mm abdominalincision and soaked with prewarmed saline. Ten ul of study substancewill be injected into each amniotic sac. The abdominal wall is thenclosed in two layers. Dams may be recovered with water and food ad lib,and pain medication.

To evaluate the development of chorioamnionitis in this model, at e17.5one can obtain the following: 1) Quantitative Blood Culture and PCR forUreaplasma: Fetal blood is obtained and quantitative PCR and culture forUreaplasma is performed. In the latter, blood is immediately incubatedin 10B broth at 37° C. in serial dilutions and color change of mediawill signal Ureaplasma growth, which is confirmed by visualization ofcolonies on agar and with PCR. (Walls et al., 2009). In the former,serum is separated and frozen for batch analysis of Ureaplasma PCR aspreviously reported. (Weisman et al., 2009) b) Quantitative AmnioticFluid Culture and PCR: Amniotic fluid is aspirated and quantitativeculture and PCR is immediately performed as described for blood. c)Quantitative Placenta Culture and PCR: Placental tissue is obtained andimmediately ground, and quantitative culture and PCR is immediatelyperformed as described for blood. d) Placenta Histopathology: Placentaltissue is obtained and immediately processed as previously described.(Redline t al., 1998) All specimens are read by a placenta pathologistblinded to the group assignment. Histologic chorioamnionitis isseparated into maternal and fetal response and assigned a stageaccordingly. (Redline et al., 1998) e) Blood, Amniotic Fluid andPlacenta Inflammatory Mediator Levels: Serum, amniotic fluid, and groundplacenta are immediately frozen in liquid nitrogen and then stored at−80° C. until a batch ELISA assay (Normann et al., 2009) forinflammatory mediators IL1α, 1L1β; IL6, TNFα, IFNγ, MIP-2, MCP-1, andTGF-β1 is performed.

To determine the effect of Ureaplasma chorioamnionitis on braindevelopment, one can use the model above and determine the following: 1)Brain Infection and Inflammation: To describe the associated braininfection and inflammation in this model, at e17.5, and 6 and 18 wksafter birth we will remove the pup's head with a guillotine, peel offthe skull and obtain: a) Quantitative Brain Culture and PCR forUreaplasma: The left hemisphere is isolated, immediately ground up andprocessed for culture and PCR as described above. b) Brain InflammatoryMediator Levels: The right hemisphere is isolated and immediately groundup, frozen in liquid nitrogen and then stored at −80° C. until a batchELISA assay is performed as described above. 2) Brain Histology andHistochemistry: To describe the associated brain pathology in thismodel, at e17.5, and 6 and 18 wks after birth one can remove the pup'shead with a guillotine, peel off the skull, place the entire brain informalin and perform routine histopathological studies. Initialcharacterization involves basic histopathology studies looking forchanges such as cortical thickness, sign of tissue loss, microcephaly,etc., for example. If pathologic, neurologic or developmental phenotypesare detected (see below) one can expand the analysis using specificneuronal markers. For evidence of synapse disease, one can use vesicularglutamate transporter to label excitatory synapses. For evidence ofgliosis one can use antibodies to glial fibrillary acidic protein(GFAP). 3) Neurologic Phenotype: To determine if a neurologic phenotypeoccurs, pups will be examined at birth, weekly for 3 weeks, then every 3wks for 18 wks to include: weight, survival, hair condition, eyecondition, spine condition, tremor, stereotypes, hind-limb clasping, andmyoclonus. These tests should provide a timeline of onset of neurologicsymptoms. Most of the evaluation can consist of observation of theanimals in the cage and in the palm of the examiner. 4) DevelopmentalPhenotype: To determine if a developmental phenotype occurs, pups canundergo the same battery of tests in the exact order listed below at 6and 18 wks after birth. These tests were selected because they arerobust and assess multiple neurobiological phenotypes including motorfunction, activity, balance and coordination, anxiety, socialinteractions, learning and memory, and abnormal movements. Developmentaltests are only performed at 18 weeks if a neurologic phenotype isobserved by 18 wks or a developmental phenotype is observed at 6 weeks.All tests are performed by investigators blinded to the groupassignment. Dowell Test: (Samaco et al., 2008) This tests coordinationand balance by placing an animal on top of a 0.7 cm horizontal dowelsuspended 60 cm above a padded surface. The time to fall is recorded.The test ends after 120 seconds. Wire Hang Test: (Samaco et al., 2008) Astring is suspended 60 cm above a padded surface and the mouse isallowed to hang onto the string by their front paws. The time to fall isrecorded. The task ends after 60 seconds. Open Field Analysis: (Samacoet al., 2008; Spencer et al., 2005) This test measures locomotoractivity and anxiety. The apparatus consists of a 40 cm×40 cm×30 cmplexiglass enclosure where an observer records the horizontal andvertical activity of the mouse. A mouse is placed inside the enclosureand monitored for 30 minutes to assess locomotion and anxiety. The totaldistance traveled and the amount of time spent moving determines amountof locomotion. The ratio of the distance traveled in the center of thefield to the total distance traveled indicates level of anxiety; animalsthat are anxious avoid the center of the field. Vertical activity isalso an indirect measure of anxiety. Light/Dark Box: (Spencer et al.,2006) The light/dark test measures anxiety based on the percentage oftime the test animals spend within the dark side of the box. Aplexiglass chamber is divided into two compartments connected by a smallopening. The “light side” compartment is made of clear Plexiglass andthe “dark side” compartment is of opaque dark plexiglass. Theenvironment is controlled with 50 lux ambient lighting and 60 dB whitenoise. The animal is placed into the anxiety-generating “light side” andthe number of transitions between sides and total time spent in eachside is recorded for 10 minutes. Total number of transitions, time spentin the light side, latency to enter the dark, and latency to enter thelight will be compared between groups. Partition Test: (Samaco et al.,2008; Spencer et al., 2005) This test measures social interaction andbehavior. The test apparatus consists of a standard cage divided in halfby a clear perforated partition. Experimental animals are individuallyhoused in one side for 3 days until eighteen hours prior to theexperiment when a gender/age/weight matched FVB:C57BL6 Fl mouse isplaced in the opposite side. An observer will be used to measure theexperimental mouse's approaches and time spent at the partition. Thefirst phase of the test measures interaction with a familiar mouse(placed eighteen hours prior to start) and the second phase measures theinteraction with a novel mouse. At the end of the test the novel mouseis replaced with the original familiar mouse and the experimental mousebehavior is scored. Morris Maze: (Watase et al., 2007) This testassesses contextual (hippocampus) and cue-based (amygdala andhippocampus) learning. Mice are trained in the Morris water maze tolocate a hidden platform. Each mouse is given four trials per day forfive consecutive days. After trial 20, each animal is given a probetrial. During the probe trial, the platform is removed and each animalis allowed to search the pool for 60 s. Tremor: (Alvarez-Saavedra etal., 2007) The degree of tremor present at 6 and 18 weeks of life willbe measured by physical examination.

To pilot if antenatal maternal treatment affects Ureaplasmachorioamnionitis related brain changes, neurological and developmentalphenotype experiments above are repeated in pups of Ureaplasma infecteddams who received our Ureaplasma rDNA vaccine prior to conception vsnon-vaccinated dams.

Sample size: Two litters (one saline and one Ureaplasma) are utilizedfor each of the blood, amniotic fluid, and placenta culture, PCR, andpathology experiments. These experiments are to describe infection andso no sample size is calculated, but the smallest sample size possibleis a litter per group. Four litters (two saline and two Ureaplasma) areutilized for the inflammatory mediator data experiments. The sample sizefor inflammatory mediator data is based on differences previouslypublished for a similar model. (Normann et al., 2009) Two litters (onesaline and one Ureaplasma) are utilized for each of the brain culture,brain PCR, and brain pathology experiments at 07.5 days, because onelitter is the smallest sample size one can select. Four litters (twosaline and two Ureaplasma) are utilized for the inflammatory mediatorexperiment at e17.5 days. The sample size for inflammatory mediator datawas based on differences previously published for a similar model.(Normann et al., 2009) The brain culture, brain PCR, brain pathologyexperiments at 6 and 18 wks, one can use about 3 pups per each timepoint/group/test or about a total 6 litters. These experiments are todescribe infection and pathology so no sample size is calculated, Thebrain inflammatory mediators at 6 and 18 wks, one can use about 15 pupsper each time point/group or about 8 litters The neurologic anddevelopmental phenotype experiments utilize 8 litters or 16 pups pergroup (saline Ureaplasma with and without vaccine) which gives one apower of 0.8 (a=0.05) to detect 1 standard deviation difference betweenthe saline and Ureaplasma groups in the unvaccinated dams and then againbetween the Ureaplasma groups of the vaccinated and unvaccinated dams,based on previous work by others. (97) One can assume a 20% wastage orloss of pups or litters based on previous work with these animals.

Data analyses: Standard statistical analyses are employed. Forcontinuous data, the distributions of data re assessed and ANOVA isutilized for those that have a normal distribution and Kruskal-Wallisfor those with a non-normal distribution. For categorical data theFischer exact or Chi-square test is performed as appropriate.

Example 4 Ureaplasma Vaccine and Related Antibodies

The present example concerns exemplary optimization of vaccine delivery,dose, and schedule and also concerns evaluation of the immunologicalresponse to the vaccine and related antibodies.

Exemplary Methods:

A DNA vaccine was delivered as follow:

-   -   1) Vaccine preparation: pDNA containing conserved segment of MBA        DNA from Ureaplasma parvum (serotype 6) was purified and diluted        with normal saline to desired concentration (as described        above). In addition, there is some data (Pathogen specific IgA,        neutralizing antibody and animal survival on a vaccine comprised        of the constant regions from both a U. parvum serotype 1 and        serotype 6, resulting in a larger plasmid with twice the DNA.    -   2) Adult FVB mice (both female and male, age 3-4 weeks) were        vaccinated with serotype 6 DNA vaccine by intraperitoneal (IP)        at dose of 200 ug to 500 ug in 1 ml NS per injection (previously        described) or by intramuscular (IM) at dose of 100 ng and 50 ug        in 0.1 ml NS.    -   3) Frequency of injection:        -   a. 500 ug×2 with 2 wks interval, IP        -   b. 500 ug×3 with 2 wks interval, IP        -   c. 200 ug×3 with 2 wks interval. IP        -   d. 50 ug×3 with 2 wks interval, IM        -   e. 100 ng×3 with 2 wks interval, IM        -   f. All animals were boosted with same amount of pDNA as the            initial inject at 12 wks after the first injection. Each            group contains 5 animals.

Results:

1. Serum Immunoglobulin Levels of Vaccinated and Control Mice:

Total IgA, IgM, IgG and Subclass IgG1, IgG2a, IgG2b, IgG2c, and IgG3were evaluated with commercially available ELISA kits. The procedurefollowed the protocol provided in the kit. The results are shown asfollow.

-   -   a. The Total IgA level is significantly increased between        vaccinated and control mouse serum, p=0.005, however there is no        statistical difference between 50 ug IM and 500 ug IP vaccine        groups. (see FIG. 1, which shows serum IgA level in vaccinated        mice)    -   b. There are no significant differences of IgM among vaccinated        and control mouse serum. p=0.5 (see FIG. 2, which shows serum        level of IgM in vaccinated mice)    -   c. The Total IgG level is significantly increased between        vaccinated and control mouse serum, p=0.008, however there is no        statistical difference between 50 ug IM and 500 ug IP vaccine        groups. The IgG subclass level is significantly increased        between vaccinated and control mouse serum for IgG1 (p=0.005),        IgG2A (p=0.0002), IgG2B (p=0.001), IgG3 (p=0.0006), but not        IgG2C (p=0.4). There is no statistical difference between the 50        ug IM and 500 ug IP vaccine groups for IgG 2C (p=0.52) and IgG 3        (p=0.17). However, IgG1 is significantly less (p=0.03) for the        50 vs. 500 ug dose, while IgG 2A (p=0.01) and IgG 2B (p=0.02)        are significantly greater for the 50 vs. 500 ug dose. (see FIG.        3, which shows serum level of IgG subclasses in vaccinated mice)

2. Serum Pathogen-Specific IgG Levels of Vaccinated and Control Mice:

Serum level of IgG against Ureaplasma parvum (serotype 14) as detectedby whole bug ELISA. The pathogen specific antibody is significantlyincreased between all the vaccine groups and controls (normal mice). Theresults are shown in FIG. 4. Note: All serum are diluted 1:2 prior toassay.

TABLE 1 Serum Level of Antibody of Mice Vaccinated with U. parvum(serotype 6) by ELISA, OD 500 500 200 50 100 Normal Mice ug X2 ug x3ugx3 ug x3 ng x3  2 wks 0.372 1.584 2.06 1.73 1.24  4 wks 0.372 1.3782.374 1.515 3.67 1.235  6 wks 0.372 1.294 1.874 2.439 0.955  8 wks 0.3721.992 1.956 10 wks 0.372 2.208 1.956 2.51 2.31 1.202 12 wks 0.372 2.6961.652 1.77 1.569 1.344 16 wks 0.372 3.432 3.78 2.402 20 wks 2.721 0.88231 wks 0.167 3.8 0.937 46 wks 2.61 0.622

3. Serum Pathogen-Specific IgA Levels of Vaccinated and Control Mice:

Serum level of IgA against Ureaplasma parvum (serotype 14) as detectedby whole bug ELISA. The results are shown in FIG. 5.

4. Serum Neutralizing (Bacterial Killing) Antibody Levels of Vaccinatedand Control Mice:

This assay was carried out on 96 cell culture plate. Each well contains:10B medium; 10² ccu Ureaplasma; serum from vaccinated or normal mice atdifferent dilutions. The plate was incubated at 37° C. for 5 days.Ureaplasma parvum serotype 1 and 6, Ureaplasma urealyticum serotype 8,and Ureaplasma diversum serotype A were used for this in vitro assay.The serum from vaccinated mice has killing activity against allUreaplasma species tested. In previous filing we have bacterial killingagainst Ureaplasma parvum serotype 14. Yellow color indicates nobacterial growth. Some of the results are shown in FIG. 6.

5. Animal Survival Following Infection of Vaccinated and Control Mice:

At 12 wks after the first injection of pDNA, all the animals received abooster injection. The mating was set up 2 days later. The pups fromthese females are infected with 2 doses of 10⁶ ccu of Ureaplasma at day1 of life (4 hrs apart). The survival rate was calculated over the next8 days and compared with pups of unvaccinated dams infected with samedose and strain of Ureaplasma. The survival rate of pups in thevaccinated group is significantly higher than the control group forevery strain of infecting Ureaplasma. However, there does not appear tobe a significant difference among the vaccinated groups in the dosestested to date. The data is displayed in FIGS. 8, 9, 10, 11 and 12.Animal survival studies are completed on the doses of 1000, 500 and 200ug per dose. Survival studies are performed on 50 ug per dose and plan100 ng per dose in the future.

In some embodiments, one can develop the protein product of the vaccineand this allows one to investigate the effects of: a protein vaccine;using the protein as a boost to the DNA vaccine (protein boost);developing and characterizing a monoclonal antibody from the protein.One can examine the effectiveness of a monoclonal antibody as atherapeutic target in humans, animals, and media/cell lines. One canimprove the delivery system/platform/method for the pDNA vaccine, forexample by investigating subcutaneous (SC) delivery at an optimum dose.One can also develop and if necessary collaborate with others to developa platform to enhance vaccine delivery IM or SC. One can develop aplatform to deliver the vaccine orally or nasally (e.g. cytofectin), forexample. One can test the vaccine utilizing an electroporation deliverysystem, for example. One can characterize the mechanism of action of thevaccine including, for example, the cellular mediated immune response.One can characterize the impact of the vaccine (pDNA or protein or both)on animal models of chronic lung disease (BPD), chorioamnionitis,vaginitis, chronic prostatitis, neurologic disorders, preterm labor,etc.

REFERENCES

All patents and publications mentioned in the specifications areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

PATENTS AND PATENT APPLICATIONS

-   U.S. Pat. No. 3,826,364-   U.S. Pat. No. 4,284,412-   U.S. Pat. No. 4,498,766-   U.S. Pat. No. 4,578,770-   U.S. Pat. No. 4,596,792-   U.S. Pat. No. 4,599,230-   U.S. Pat. No. 4,599,231-   U.S. Pat. No. 4,601,903-   U.S. Pat. No. 4,608,251-   U.S. Pat. No. 4,661,913-   U.S. Pat. No. 4,774,189-   U.S. Pat. No. 4,767,206-   U.S. Pat. No. 4,714,682-   U.S. Pat. No. 4,857,451-   U.S. Pat. No. 4,989,977-   U.S. Pat. No. 5,160,974-   U.S. Pat. No. 5,478,722

PUBLICATIONS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of inhibiting or reducing symptoms ofUreaplasma infection in an individual, comprising the step of deliveringto the individual a therapeutically effective amount of antibodies thatrecognize the conserved region of Ureaplasma multiple-banded antigen orthe 5′ end of the multiple-banded antigen.
 2. The method of claim 1,further comprising multiple steps of delivering antibodies to theindividual.
 3. The method of claim 2, wherein the multiple deliveriesare separated by one month or more.
 4. The method of claim 2, whereinthe multiple deliveries are separated by days or weeks.
 5. The method ofclaim 2, wherein the multiple deliveries are separated by one year, twoyears, or ten years.
 6. The method of claim 1, wherein the delivery isinto the amniotic cavity.
 7. The method of claim 1, wherein the deliveryof antibodies is vaginally administered.
 8. The method of claim 1,wherein the individual is a female or male prior to a first sexualactivity.
 9. The method of claim 1, wherein the individual is a femaleprior to pregnancy.
 10. The method of claim 1, wherein the individual isa pregnant female.
 11. The method of claim 1, wherein the antibodiesrecognize the 5′ end of the Ureaplasma multiple-banded antigen.
 12. Themethod of claim 1, wherein the antibodies recognize the Ureaplasmasequence of SEQ ID No:
 4. 13. The method of claim 1, wherein theindividual is of one of the species human, cow, dog, cat, horse, pig,goat, sheep, or bird.